U.S. patent application number 13/957679 was filed with the patent office on 2013-12-05 for purified antibody composition.
This patent application is currently assigned to ABBVIE BIOTECHNOLOGY LTD.. The applicant listed for this patent is George Avgerinos, Min Wan, Gregory Zarbis-Papastoitsis. Invention is credited to George Avgerinos, Min Wan, Gregory Zarbis-Papastoitsis.
Application Number | 20130323261 13/957679 |
Document ID | / |
Family ID | 38581592 |
Filed Date | 2013-12-05 |
United States Patent
Application |
20130323261 |
Kind Code |
A1 |
Wan; Min ; et al. |
December 5, 2013 |
PURIFIED ANTIBODY COMPOSITION
Abstract
The invention provides a method for producing a host cell
protein-(HCP) reduced antibody preparation from a mixture
comprising an antibody and at least one HCP, comprising an ion
exchange separation step wherein the mixture is subjected to a
first ion exchange material, such that the HCP-reduced antibody
preparation is obtained.
Inventors: |
Wan; Min; (Worcester,
MA) ; Avgerinos; George; (Sudbury, MA) ;
Zarbis-Papastoitsis; Gregory; (Watertown, MA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Wan; Min
Avgerinos; George
Zarbis-Papastoitsis; Gregory |
Worcester
Sudbury
Watertown |
MA
MA
MA |
US
US
US |
|
|
Assignee: |
; ABBVIE BIOTECHNOLOGY LTD.
Hamilton
BM
|
Family ID: |
38581592 |
Appl. No.: |
13/957679 |
Filed: |
August 2, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13532511 |
Jun 25, 2012 |
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13957679 |
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12882601 |
Sep 15, 2010 |
8231876 |
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13532511 |
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11732918 |
Apr 4, 2007 |
7863426 |
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12882601 |
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60789725 |
Apr 5, 2006 |
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60790414 |
Apr 6, 2006 |
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Current U.S.
Class: |
424/142.1 ;
424/158.1 |
Current CPC
Class: |
A61K 39/3955 20130101;
A61P 37/00 20180101; A61P 17/06 20180101; A61P 31/14 20180101; A61P
19/02 20180101; C07K 1/36 20130101; A61K 47/22 20130101; A61P 29/02
20180101; A61P 31/00 20180101; A61K 47/26 20130101; A61P 3/00
20180101; A61P 11/00 20180101; A61K 31/519 20130101; C07K 1/18
20130101; C07K 16/065 20130101; C07K 16/241 20130101; A61P 13/12
20180101; A61P 1/04 20180101; A61P 3/10 20180101; A61P 9/00
20180101; A61P 37/06 20180101; A61P 37/08 20180101; C07K 2317/14
20130101; A61K 2039/54 20130101; A61P 19/06 20180101; A61P 1/00
20180101; C07K 2317/21 20130101; A61P 17/00 20180101; A61P 27/02
20180101; A61P 25/00 20180101; A61P 9/10 20180101; A61P 35/00
20180101; A61K 2039/505 20130101; A61P 3/04 20180101; C07K 2317/94
20130101; A61P 29/00 20180101; A61P 43/00 20180101; A61K 2039/545
20130101; A61K 39/39591 20130101 |
Class at
Publication: |
424/142.1 ;
424/158.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A method of treating a disorder in which TNF.alpha. activity is
detrimental in a subject, the method comprising administering a
composition comprising an isolated human anti-TNF.alpha. antibody
to the subject, such that the disorder is treated, wherein the
composition has a cathepsin L activity of less than 1.84 RFU/s/mg
antibody, and wherein the human anti-TNF.alpha. antibody comprises
a light chain variable region comprising a light chain CDR3 domain
comprising the amino acid sequence of SEQ ID NO: 3; a light chain
CDR2 domain comprising the amino acid sequence of SEQ ID NO: 5; and
a light chain CDR1 domain comprising the amino acid sequence of SEQ
ID NO: 7; and comprises a heavy chain variable reason comprising a
heavy chain CDR3 domain comprising the amino acid sequence of SEQ
ID NO: 4; a heavy chain CDR2 domain comprising the amino acid
sequence of SEQ ID NO: 6; and a heavy chain CDR1 domain comprising
the amino acid sequence of SEQ ID NO: 8.
2. The method of claim 1, wherein the composition has a cathepsin L
activity of no greater than 1.3 RFU/s/mg antibody.
3. The method of claim 1, wherein the composition has a cathepsin L
activity of no greater than 0.6 RFU/s/mg antibody.
4. The method of claim 1, wherein the composition has a cathepsin L
activity of 0.6 to less than 1.84 RFU/s/mg antibody.
5. The method of claim 1, wherein the disorder is selected from the
group consisting of rheumatoid arthritis, Crohn's disease,
ulcerative colitis, ankylosing spondylitis, psoriatic arthritis,
psoriasis, and juvenile rheumatoid arthritis.
6. A method of treating a disorder in which TNF.alpha. activity is
detrimental in a subject, the method comprising administering a
composition comprising an isolated human anti-TNF.alpha. antibody
to the subject, such that the disorder is treated, wherein the
composition has a cathepsin L activity of less than 1.84 RFU/s/mg
antibody, and wherein the human anti-TNF.alpha. antibody comprises
a light chain variable region (LCVR) comprising the amino acid
sequence of SEQ ID NO: 1, and a heavy chain variable region (HCVR)
comprising the amino acid sequence of SEQ ID NO: 2.
7. The method of claim 6, wherein the composition has a cathepsin L
activity of no greater than 1.3 RFU/s/mg antibody.
8. The method of claim 6, wherein the composition has a cathepsin L
activity of no greater than 0.6 RFU/s/mg antibody.
9. The method of claim 6, wherein the composition has a cathepsin L
activity of 0.6 to less than 1.84 RFU/s/mg antibody.
10. The method of claim 6, wherein the disorder is selected from
the group consisting of rheumatoid arthritis, Crohn's disease,
ulcerative colitis, ankylosing spondylitis, psoriatic arthritis,
psoriasis, and juvenile rheumatoid arthritis.
11. A method of treating a disorder in which TNF.alpha. activity is
detrimental in a subject, the method comprising administering a
composition comprising an isolated human anti-TNF.alpha. antibody
to the subject, such that the disorder is treated, wherein the
composition has a cathepsin L activity of less than 1.84 RFU/s/mg
antibody, and wherein the antibody is adalimumab.
12. The method of claim 11, wherein the composition has a cathepsin
L activity of no greater than 1.3 RFU/s/mg antibody.
13. The method of claim 11, wherein the composition has a cathepsin
L activity of no greater than 0.6 RFU/s/mg antibody.
14. The method of claim 11, wherein the composition has a cathepsin
L activity of 0.6 to less than 1.84 RFU/s/mg antibody.
15. The method of claim 11, wherein the composition comprises a
cathepsin L activity ranging from between 0.6 to 1.3 RFU/s/mg
antibody.
16. The method of claim 11, wherein the disorder is selected from
the group consisting of rheumatoid arthritis, Crohn's disease,
ulcerative colitis, ankylosing spondylitis, psoriatic arthritis,
psoriasis, and juvenile rheumatoid arthritis.
17. The method of claim 1, wherein the antibody comprises a heavy
chain IgG.sub.1 constant region.
18. The method of claim 17, wherein the antibody comprises a kappa
light chain constant region.
19. The method of claim 1, wherein the antibody is produced by a
mammalian expression system.
20. The method of claim 19, wherein the mammalian expression system
is a Chinese Hamster Ovary (CHO) expression system.
21. The method of claim 20, wherein the disorder is selected from
the group consisting of rheumatoid arthritis, Crohn's disease,
ulcerative colitis, ankylosing spondylitis, psoriatic arthritis,
psoriasis, and juvenile rheumatoid arthritis.
22. A method of treating a disorder in which TNF.alpha. activity is
detrimental in a subject, the method comprising subcutaneously
administering a composition comprising an isolated human
anti-TNF.alpha. antibody to the subject, such that the disorder is
treated, wherein the composition has a cathepsin L activity of 0.6
to less than 1.84 RFU/s/mg antibody; wherein the human
anti-TNF.alpha. antibody comprises a light chain variable region
comprising a light chain CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 3; a light chain CDR2 domain comprising the
amino acid sequence of SEQ ID NO: 5; and a light chain CDR1 domain
comprising the amino acid sequence of SEQ ID NO: 7; and comprises a
heavy chain variable reason comprising a heavy chain CDR3 domain
comprising the amino acid sequence of SEQ ID NO: 4; a heavy chain
CDR2 domain comprising the amino acid sequence of SEQ ID NO: 6; and
a heavy chain CDR1 domain comprising the amino acid sequence of SEQ
ID NO: 8; and wherein the disorder is selected from the group
consisting of rheumatoid arthritis, Crohn's disease, ulcerative
colitis, ankylosing spondylitis, psoriatic arthritis, psoriasis,
and juvenile rheumatoid arthritis
23. The method of any claim 22, wherein the human anti-TNF.alpha.
antibody comprises a light chain variable region (LCVR) comprising
the amino acid sequence of SEQ ID NO: 1, and a heavy chain variable
region (HCVR) comprising the amino acid sequence of SEQ ID NO:
2.
24. The method of claim 22, wherein the human anti-TNF.alpha.
antibody is adalimumab.
25. The method of claim 22, wherein the antibody is produced by a
mammalian expression system.
26. The method of claim 22, wherein the mammalian expression system
is a Chinese Hamster Ovary (CHO) expression system.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 13/532,511, filed on Jun. 25, 2012, which, in
turn, is a continuation of U.S. patent application Ser. No.
12/882,601, filed on Sep. 15, 2010, now issued as U.S. Pat. No.
8,231,876; which is a divisional of U.S. patent application Ser.
No. 11/732,918, filed on Apr. 4, 2007, now issued as U.S. Pat. No.
7,863,426; which claims priority to U.S. provisional application
Ser. No. 60/789,725, filed on Apr. 5, 2006 and to U.S. provisional
application Ser. No. 60/790,414, filed on Apr. 6, 2006, the
contents of each of which are hereby incorporated in their
entirety.
BACKGROUND OF THE INVENTION
[0002] Large-scale, economic purification of proteins is
increasingly an important problem for the biotechnology industry.
Generally, proteins are produced by cell culture, using either
mammalian or bacterial cell lines engineered to produce the protein
of interest by insertion of a recombinant plasmid comprising the
gene for that protein. Since the cell lines used are living
organisms, they must be fed with a complex growth medium,
comprising sugars, amino acids, and growth factors, usually
supplied from preparations of animal serum. Separation of the
desired protein from the mixture of compounds fed to the cells and
from the by-products of the cells themselves to a purity sufficient
for use as a human therapeutic poses a formidable challenge.
SUMMARY OF THE INVENTION
[0003] There is a need for improved methods of obtaining antibody
preparations comprising a reduced amount of host cell protein,
including procathepsin L. The invention provides a method for
purifying antibodies expressed in a host cell expression system,
wherein the resulting antibody preparation comprises a reduced
amount of host cell protein, including procathepsin L. The improved
method of the invention also includes the development of
reproducible methods of accurately detecting host cell proteins,
and a kinetic assay
[0004] The invention provides a method for producing a host cell
protein-(HCP) reduced antibody preparation from a mixture
comprising an antibody and at least one HCP, comprising an ion
exchange separation step wherein the mixture is subjected to a
first ion exchange material, such that the HCP-reduced antibody
preparation is obtained. In one embodiment, the ion exchange
separation step comprises passing the mixture over the first ion
exchange material such that a first eluate having a reduced level
of HCP is obtained. In one embodiment, the method of the invention
further comprises a second ion exchange separation step wherein the
first eluate is subjected to a second ion exchange material such
that a first flowthrough having a reduced level of HCP is obtained.
In another embodiment, the method of the invention further
comprises a hydrophobic interaction separation step wherein the
first flowthrough is subjected to a first hydrophobic interaction
material such that a second eluate having a reduced level of HCP is
obtained.
[0005] In one embodiment of the invention, the ion exchange
separation step comprises a first ion exchange chromatography step,
wherein the mixture is loaded onto a column comprising the first
ion exchange material, such that a first eluate having a reduced
level of HCP is obtained. In one embodiment, the invention further
comprises a second ion exchange chromatography step comprising
loading the first eluate onto a column comprising a second ion
exchange material, such that a first flowthrough is obtained.
[0006] In one embodiment, the invention further comprises a
hydrophobic interaction separation step comprising loading the
first flowthrough onto a column comprising a first hydrophobic
interaction material, such that a second eluate is obtained. In one
embodiment, the hydrophobic interaction separation step comprises
hydrophobic interaction chromatography. In one embodiment, the
hydrophobic interaction chromatography is phenyl sepharose
chromatography. In still another embodiment, the amount of antibody
loaded on to the hydrophobic interaction material ranges from about
20 to about 40 grams of antibody per liter of hydrophobic
interaction material. In yet another embodiment, the amount of
antibody loaded on to the hydrophobic interaction material ranges
from about 30 to about 36 grams of antibody per liter of
hydrophobic interaction material.
[0007] In one embodiment, the ion exchange chromatography step is
cation exchange chromatography. In another embodiment, the cation
exchange chromatography is a synthetic methacrylate based polymeric
resin attached to a sulfonate group. In still another embodiment,
the invention further comprises washing the ion exchange material
with a plurality of wash steps. In one embodiment, the plurality of
wash steps comprises an increase in conductivity. In one
embodiment, the ion exchange material is washed with a wash
comprising about 40-50% elution buffer and about 50-60% water
(e.g., water for injection (WFI)). In one embodiment, the elution
buffer is 20 mM Na.sub.2PO.sub.4, 150 mM sodium chloride, pH 7.
[0008] In one embodiment, the first eluate is subjected to viral
inactivation prior to the first ion exchange chromatography step.
In one embodiment, the viral inactivation is achieved through pH
viral inactivation (e.g., lower the pH of the first eluate to
thereby inactivate viruses).
[0009] In one embodiment of the invention, the second ion exchange
chromatography step comprises anion exchange chromatography. In one
embodiment, the anion exchange chromatography is Q sepharose
chromatography.
[0010] The invention also provides a method method for producing a
host cell protein-(HCP) reduced antibody preparation from a mixture
comprising an antibody and at least one HCP, wherein the reduced
level of HCP is achieved by altering the pH and conductivity of the
first eluate such that the pH and conductivity of the first eluate
is substantially similar to the pH and conductivity of the second
ion exchange material. In one embodiment, the pH of the second ion
exchange material ranges from about 7.7 to about 8.3. In another
embodiment, the pH of the first eluate is altered to range from
about 7.7 to about 8.3. In still another embodiment, the pH of the
first eluate is altered to about 8.0. In one embodiment, the
conductivity of the second ion exchange material ranges from about
3.5 mS/cm to about 5.2 mS/cm, or from about 3.5 mS/cm to about 4.9
mS/cm. In one embodiment, the conductivity of the first eluate is
altered to range from about 3.5 mS/cm to about 5.2 mS/cm or from
about 3.5 mS/cm to about 4.9 mS/cm.
[0011] In one embodiment of the invention, the first eluate
comprises a range of about 90 to about 100 fold less HCP than the
mixture as determined by a HCP ELISA. In another embodiment, the
first flowthrough comprises a range of about 840 to about 850 fold
less HCP than the first eluate as determined by a HCP ELISA. In yet
another embodiment, the second eluate comprises a range of about 3
to about 5 fold less HCP than the first flowthrough as determined
by a HCP ELISA.
[0012] The invention provides a method for producing a procathepsin
L-reduced antibody preparation from a mixture comprising an
antibody and procathepsin L, comprising an ion exchange separation
step wherein the mixture is subjected to a first ion exchange
material, such that the procathepsin L-reduced antibody preparation
is obtained.
[0013] In one embodiment, the ion exchange separation step
comprises passing the mixture over the first ion exchange material
such that a first eluate having a reduced level of procathepsin L
is obtained. In one embodiment, the ion exchange separation step
comprises a first ion exchange chromatography step, wherein the
mixture is loaded onto a column comprising the first ion exchange
material, such that a first eluate having a reduced level of
procathepsin L is obtained.
[0014] In one embodiment, the invention further comprises a second
ion exchange separation step wherein the first eluate is subjected
to a second ion exchange material such that a first flowthrough
having a reduced level of procathepsin L is obtained. In one
embodiment, the invention further comprises a second ion exchange
chromatography step comprising loading the first eluate onto a
column comprising a second ion exchange material, such that a first
flowthrough is obtained.
[0015] In one embodiment, the invention further comprises a
hydrophobic interaction separation step wherein the first
flowthrough is subjected to a first hydrophobic interaction
material such that a second eluate having a reduced level of
procathepsin L is obtained. In another embodiment, the invention
further comprises a hydrophobic interaction separation step
comprising loading the first flowthrough onto a column comprising a
first hydrophobic interaction material, such that a second eluate
is obtained.
[0016] In one embodiment, the ion exchange chromatography step is
cation exchange chromatography, including, but not limited to a
synthetic methacrylate based polymeric resin attached to a
sulfonate group.
[0017] In another embodiment, the ion exchange chromatography step
further comprises washing the ion exchange material with a
plurality of wash steps. In one embodiment, the plurality of wash
steps comprises an increase in conductivity. In one embodiment, the
ion exchange material is washed with a wash buffer comprising about
40-50% elution buffer and about 50-60% water (e.g., water for
injection (WFI)). In still another embodiment, the elution buffer
is 20 mM Na.sub.2PO.sub.4, 150 mM sodium chloride, pH 7.
[0018] In one embodiment of the invention, the first eluate is
subjected to viral inactivation prior to ion exchange
chromatography step. In one embodiment, the viral inactivation is
achieved through pH viral inactivation (e.g., lowering of the pH of
the first eluate to thereby inactive viruses).
[0019] In one embodiment, the ion exchange chromatography step
comprises anion exchange chromatography. In one embodiment, the
anion exchange chromatography is Q sepharose chromatography.
[0020] The invention also describes a method wherein the reduced
level of procathepsin L is attained by altering the pH and
conductivity of the first eluate such that the pH and conductivity
of the first eluate is substantially similar to the pH and
conductivity of the second ion exchange material. In one
embodiment, the pH of the second ion exchange material ranges from
about 7.7 to about 8.3. In another embodiment the pH of the first
eluate is altered to range from about 7.7 to about 8.3. In still
another embodiment wherein the pH of the first eluate is altered to
about 8.0. In yet another embodiment, the conductivity of the
second ion exchange material ranges from about 3.5 mS/cm to about
5.2 mS/cm, or from about 3.5 mS/cm to about 4.9 mS/cm. In one
embodiment, the conductivity of the first eluate is altered to
range from about 3.5 mS/cm to about 5.2 mS/cm, or from about 3.5
mS/cm to about 4.9 mS/cm.
[0021] In one embodiment of the invention, the hydrophobic
interaction separation step comprises hydrophobic interaction
chromatography. In one embodiment, the hydrophobic interaction
chromatography is phenyl sepharose chromatography. In still another
embodiment, the amount of antibody loaded on to the hydrophobic
interaction material ranges from about 20 to about 40 grams of
antibody per liter of hydrophobic interaction material. In yet
another embodiment, the amount of antibody loaded on to the
hydrophobic interaction material ranges from about 30 to about 36
grams of antibody per liter of hydrophobic interaction
material.
[0022] In one embodiment, the first eluate comprises cathepsin L
activity ranging from between about 25 to about 60 RFU/s/mg of
antibody as measured by a cathepsin L kinetic assay.
[0023] In another embodiment, the first flowthrough comprises
cathepsin L activity ranging from between about 0.4 to about 4
RFU/s/mg of antibody as measured by a cathepsin L kinetic
assay.
[0024] In still another embodiment, the second eluate comprises
cathepsin L activity ranging from between about 0.5 to about 1.5
RFU/s/mg of antibody as measured by a cathepsin L kinetic
assay.
[0025] In one embodiment of the invention, the level of
procathepsin L is reproducibly low.
[0026] In a particularly preferred aspect, the invention provides
antibody purification methods in which high amounts of an
antibody-HCP mixture can be loaded onto an ion exchange resin to
achieve reduction in HCP in the mixture. This methodology has the
advantage that it can be used with antibody-HCP mixtures that have
not been subjected to protein A capture prior to application of the
antibody-HCP mixture to the ion exchange resin. Protein A capture,
in which an antibody-HCP mixture is applied to a protein A column
such that the antibody binds to protein A and HCPs flow through,
typically is used as an initial purification step in antibody
purification procedures as a means to remove HCPs. Thus, the
methods of the invention are useful for purifying large loads of
antibody-HCP mixtures without the need to carry out a protein A
chromatography as an initial step.
[0027] Thus, in one embodiment, the invention provides a method for
producing a host cell protein-(HCP) reduced antibody preparation
from a mixture comprising an antibody and at least one HCP, the
method comprising:
[0028] (a) applying the mixture to a first ion exchange resin in an
equilibration buffer, wherein greater than 30 grams of antibody per
liter of resin are applied;
[0029] (b) washing HCP from the resin with a plurality of wash
steps; and
[0030] (c) eluting the antibody from the resin with an elution
buffer to form a first eluate,
[0031] such that the HCP-reduced antibody preparation is
obtained.
In another embodiment, about 35-70 grams of antibody per liter of
resin are applied. In yet another embodiment, about 70 grams of
antibody per liter of resin are applied. In a preferred embodiment,
the mixture comprising an antibody and at least one HCP is not
subjected to protein A capture (e.g., is not applied to protein A
column) prior to applying the mixture to the first ion exchange
resin.
[0032] Preferably, the plurality of wash steps comprises at least a
first wash and a second wash, wherein there is an increase in
conductivity from the first wash to the second wash. More
preferably, the first wash is with equilibration buffer and the
second wash is with a mixture of elution buffer and water (e.g.,
WFI). For example, the mixture of elution buffer and water can
comprise about 40-50% elution buffer and about 50-60% water. More
preferably, the mixture of elution buffer and water can comprise
about 45% elution buffer and about 55% water. In a preferred
embodiment, the elution buffer comprises 20 mM sodium phosphate and
150 mM sodium chloride. In this situation, a mixture of elution
buffer and water that is 45% elution buffer and about 55% water is
9 mM sodium phosphate and 68 mM sodium chloride. In a preferred
embodiment, the first wash is with an equilibrium buffer comprising
20 mM phosphate, 25 mM sodium chloride, the second wash is with a
buffer comprising 9 mM phosphate, 68 mM sodium chloride (45%
elution buffer, 55% water) and the elution buffer comprises 20 mM
sodium phosphate and 150 mM sodium chloride.
[0033] In one embodiment, the method using the first ion exchange
resin is carried out at pH 7. In another embodiment, the method
using the first ion exchange resin is carried out at pH 5. In yet
another embodiment, the method using the first ion exchange resin
is carried out at a pH in a range of about pH 5 to about pH 7, or a
range of pH 5 to pH 7. When pH 7 is used, preferably about 35 grams
of antibody per liter of resin is applied. When pH 5 is used,
preferably about 70 grams of antibody per liter of resin is
applied. When a pH in the range of about pH 5 to about pH 7 (e.g.,
pH 5 to pH 7) is used, preferably an amount of antibody from about
35 to about 70 grams of antibody per liter of resin (e.g., 35-70
grams of antibody per liter of resin) is applied.
[0034] In a preferred embodiment, better HCP clearance from the
antibody-HCP mixture is achieved (e.g., at pH 5) by loading more
antibody onto the resin (e.g., about 70 grams of antibody per liter
of resin) than is achieved when less antibody (e.g., about 30 grams
of antibody per liter of resin) is loaded onto the resin. This is
thought to be the result of displacement of HCP from the resin by
the antibody when conditions are used at which the binding affinity
of the antibody for the resin is significantly greater than that of
HCP for the resin.
[0035] Preferably, the first ion exchange resin is a cation
exchange resin. Preferably, the cation exchange resin is formed
into a column and the mixture comprising the antibody and at least
one HCP is applied to the column. Preferably, the cation exchange
resin comprises a synthetic methacrylate based polymeric resin
attached to a sulfonate group (e.g., Fractogel S). Alternatively,
the cation exchange resin can comprise, for example, methacrylate
or polystyrene based synthetic polymers, silica, highly
cross-linked agarose with dextran surface extender, cross-linked
copolymer of allyl dextran and N. N. methylene bisacryla resins
attached to a sulfonate group, such as sulfonium ions or
sulfoethyl.
[0036] In another aspect of the invention, after the method using
the first ion exchange resin described above is carried out, the
method further comprises subjecting the first eluate to a viral
inactivation step. For example, wherein viral inactivation can be
achieved by pH viral inactivation to form a virally-inactived
preparation (e.g., the first eluate is subjected to low pH
conditions, such as pH of about 3.5, to thereby inactivate
viruses). Preferably, the virally-inactivated preparation is
applied to a second ion exchange resin, wherein, prior to applying
the virally-inactivated preparation to the second ion exchange
resin, pH and conductivity of the virally-inactivated preparation
is adjusted to be substantially similar to pH and conductivity of
the second ion exchange resin. For example, the pH of the second
ion exchange resin can be in a range of about pH 7.7 to about pH
8.3 and the pH of the virally-inactivated preparation is adjusted
to be in a range of about pH 7.7 to about pH 8.3. In another
embodiment, the pH of the second ion exchange resin can be in a
range of about pH 7.8 to about pH 8.2 and the pH of the
virally-inactivated preparation is adjusted to be in a range of
about pH 7.8 to about pH 8.2. More preferably, the pH of the second
ion exchange resin is about pH 8.0 and the pH of the
virally-inactivated preparation is adjusted to be about pH 8.0.
Furthermore, the conductivity of the second ion exchange resin can
be in a range of about 3.5 mS/cm to about 5.2 mS/cm and the
conductivity of the virally-inactivated preparation is adjusted to
be in a range of about 3.5 mS/cm to about 5.2 mS/cm. Preferably,
the conductivity of the second ion exchange resin is about 5.0
mS/cm and the conductivity of the virally-inactivated preparation
is adjusted to be about 5.0 mS/cm.
[0037] In a preferred embodiment, the second ion exchange resin is
an anion exchange resin. For example, the anion exchange resin can
be a Q sepharose resin. Preferably, the second ion exchange resin
is formed into a column and the virally-inactivated preparation is
applied to the column such that a first flow through is
obtained.
[0038] In another aspect of the invention, after the first through
is obtained from the second ion exchange resin, the first flow
through can be applied to a hydrophobic interaction column such
that a second eluate is obtained. In a preferred embodiment, the
hydrophobic interaction column is a phenyl sepharose column. In one
embodiment, the first flow through applied to the hydrophobic
interaction column comprises about 20 to about 40 grams of antibody
per liter of hydrophobic interaction column material. In another
embodiment, the first flow through applied to the hydrophobic
interaction column comprises about 30 to about 36 grams of antibody
per liter of hydrophobic interaction column material. Due to the
efficiency of the prior steps in the purification process, it has
been found that it is not necessary to subject the second eluate,
obtained from the hydrophobic interaction column, to product peak
fractionation. Thus, in one embodiment, the second eluate is not
subjected to product peak fractionation.
[0039] In a particularly preferred embodiment, the method of the
invention for producing a host cell protein-(HCP) reduced antibody
preparation from a mixture comprising an antibody and at least one
HCP comprises:
[0040] (a) applying the mixture to a cation exchange resin in an
equilibration buffer, wherein the mixture has not been subjected to
protein A capture prior to applying to the cation exchange resin
and wherein greater than 30 grams of antibody per liter of resin
are applied;
[0041] (b) washing HCP from the cation exchange resin with a
plurality of wash steps;
[0042] (c) eluting the antibody from the cation exchange resin with
an elution buffer to form a first eluate;
[0043] (d) subjecting the first eluate to a viral inactivation
step;
[0044] (e) applying the virally-inactivated preparation to an anion
exchange resin to obtain a first flow through; and
[0045] (f) applying the first flow through to a hydrophobic
interaction column such that a second eluate is obtained;
[0046] such that the HCP-reduced antibody preparation is
obtained.
[0047] In one embodiment, the cation exchange resin is at pH 7 and
about 35 grams of antibody per liter of resin are applied. In
another embodiment, the cation exchange resin is at pH 5 and about
70 grams of antibody per liter of resin are applied. In yet another
embodiment, the pH is in a range of about pH 5 to about pH 7 (e.g.,
pH 5 to pH 7) and an amount of antibody from about 35 to about 70
grams of antibody per liter of resin (e.g., 35-70 grams of antibody
per liter of resin) is applied.
[0048] Preferably, the plurality of wash steps comprises washing
the resin with a first wash using the equilibration buffer and a
second wash using a mixture of the elution buffer and water. For
example, the mixture of elution buffer and water can comprise about
40-50% elution buffer and about 50-60% water (e.g., WFI), more
preferably about 45% elution buffer and about 55% water (e.g.,
WFI). In a preferred embodiment, the elution buffer comprises 20 mM
sodium phosphate and 150 mM sodium chloride. In this situation, a
mixture of elution buffer and water that is 45% elution buffer and
about 55% water is 9 mM sodium phosphate and 68 mM sodium chloride.
In a preferred embodiment, the first wash is with an equilibrium
buffer comprising 20 mM phosphate, 25 mM sodium chloride, the
second wash is with a buffer comprising 9 mM phosphate, 68 mM
sodium chloride (45% elution buffer, 55% water) and the elution
buffer comprises 20 mM sodium phosphate and 150 mM sodium
chloride.
[0049] Preferably, in the above-described method with steps (a)
through (0, prior to applying the virally-inactivated preparation
to the anion ion exchange resin (i.e., between steps (d) and (e)),
pH and conductivity of the virally-inactivated preparation is
adjusted to be substantially similar to pH and conductivity of the
anion exchange resin. For example, the pH of the second ion
exchange resin can be in a range of about pH 7.7 to about pH 8.3
and the pH of the virally-inactivated preparation is adjusted to be
in a range of about pH 7.7 to about pH 8.3. In another embodiment,
the pH of the second ion exchange resin can be in a range of about
pH 7.8 to about pH 8.2 and the pH of the virally-inactivated
preparation is adjusted to be in a range of about pH 7.8 to about
pH 8.2. More preferably, the pH of the second ion exchange resin is
about pH 8.0 and the pH of the virally-inactivated preparation is
adjusted to be about pH 8.0. Furthermore, the conductivity of the
second ion exchange resin can be in a range of about 3.5 mS/cm to
about 5.2 mS/cm and the conductivity of the virally-inactivated
preparation is adjusted to be in a range of about 3.5 mS/cm to
about 5.2 mS/cm. Preferably, the conductivity of the second ion
exchange resin is about 5.0 mS/cm and the conductivity of the
virally-inactivated preparation is adjusted to be about 5.0
mS/cm.
[0050] In the above-described method with steps (a) through (f),
preferably the cation exchange resin is a synthetic
methacrylate-based polymeric resin attached to a sulfonate group
(e.g., Fractogel), the anion exchange resin is a Q sepharose resin
and the hydrophobic interaction column is a phenyl sepharose
column.
[0051] Preferably, the first eluate comprises a range of about 90
to about 100 fold less HCP than the mixture as determined by a HCP
ELISA. Preferably, the first flowthrough comprises a range of about
840 to about 850 fold less HCP than the first eluate as determined
by a HCP ELISA. Preferably, the second eluate comprises a range of
about 3 to about 5 fold less HCP than the first flowthrough as
determined by a HCP ELISA.
[0052] In a particularly preferred embodiment, the method of the
invention for producing a host cell protein-(HCP) reduced antibody
preparation from a mixture comprising an antibody and at least one
HCP comprises:
[0053] (a) applying the mixture to a cation exchange resin in an
equilibration buffer, wherein the cation exchange resin is at pH 7
and about 35 grams of antibody per liter of resin are applied, or
the cation exchange resin is at a pH in a range of pH 5 to pH 7 and
about 35 to about 70 grams of antibody per liter of resin are
applied, or the cation exchange resin is at pH 5 and about 70 grams
of antibody per liter of resin are applied;
[0054] (b) washing HCP from the cation exchange resin with wash
steps comprising a first wash using the equilibration buffer and a
second wash using a mixture of an elution buffer and water;
[0055] (c) eluting the antibody from the cation exchange resin with
the elution buffer to form a first eluate;
[0056] (d) subjecting the first eluate to a viral inactivation
step, wherein viral inactivation is achieved by pH viral
inactivation to form a virally-inactived preparation;
[0057] (e) applying the virally-inactivated preparation to an anion
exchange resin, wherein, prior to applying the virally-inactivated
preparation to the anion ion exchange resin, pH and conductivity of
the virally-inactivated preparation is adjusted to be substantially
similar to pH and conductivity of the anion exchange resin, such
that a first flow through is obtained; and
[0058] (f) applying the first flow through to a hydrophobic
interaction column such that a second eluate is obtained;
[0059] such that the HCP-reduced antibody preparation is
obtained.
[0060] Preferably, the antibody mixture has not been subjected to
protein A capture prior to applying to the cation exchange resin.
Preferably, the mixture of elution buffer and water comprises about
40-50% elution buffer and about 50-60% water, more preferably about
45% elution buffer and about 55% water (e.g., WFI). In a preferred
embodiment, the elution buffer comprises 20 mM sodium phosphate and
150 mM sodium chloride. In this situation, a mixture of elution
buffer and water that is 45% elution buffer and about 55% water is
9 mM sodium phosphate and 68 mM sodium chloride. In a preferred
embodiment, the first wash is with an equilibrium buffer comprising
20 mM phosphate, 25 mM sodium chloride, the second wash is with a
buffer comprising 9 mM phosphate, 68 mM sodium chloride (45%
elution buffer, 55% water) and the elution buffer comprises 20 mM
sodium phosphate and 150 mM sodium chloride. Preferably, the first
eluate comprises a range of about 90 to about 100 fold less HCP
than the mixture as determined by a HCP ELISA. Preferably, the
first flowthrough comprises a range of about 840 to about 850 fold
less HCP than the first eluate as determined by a HCP ELISA.
Preferably, the second eluate comprises a range of about 3 to about
5 fold less HCP than the first flowthrough as determined by a HCP
ELISA.
[0061] In a preferred aspect of any of the above-described
purification methods, the HCP comprises procathepsin L such that a
procathepsin L-reduced antibody preparation is obtained.
Preferably, the eluate comprises cathepsin L activity ranging from
between about 25 to about 60 RFU/s/mg of antibody as measured by a
cathepsin L kinetic assay. Preferably, the first flowthrough
comprises cathepsin L activity ranging from between about 0.4 to
about 4 RFU/s/mg of antibody as measured by a cathepsin L kinetic
assay. Preferably, the second eluate comprises cathepsin L activity
ranging from between about 0.5 to about 1.5 RFU/s/mg of antibody as
measured by a cathepsin L kinetic assay Preferably, the level of
procathepsin L is reproducibly low.
[0062] In yet another aspect, the invention pertains to a method
for producing a host cell protein-(HCP) reduced antibody
preparation from a mixture comprising an antibody and at least one
HCP, the method comprising:
[0063] (a) applying the mixture to a cation exchange resin to
obtain a first eluate;
[0064] (b) applying the first eluate to an anion ion exchange resin
to obtain a first flow through; and
[0065] (c) applying the first flow through to a hydrophobic
interaction column such that a second eluate is obtained;
[0066] such that the HCP-reduced antibody preparation is
obtained.
[0067] Preferably, the mixture comprising an antibody and at least
one HCP is not subjected to protein A capture prior to applying the
mixture to the first ion exchange resin. Preferably, the method
further comprises subjecting the first eluate to a viral
inactivation step prior to applying the first eluate to the anion
exchange resin. For example, viral inactivation can be achieved by
pH viral inactivation.
[0068] Preferably, the cation exchange resin comprises a synthetic
methacrylate based polymeric resin attached to a sulfonate group
(e.g., the cation exchange resin can be a Fractogel S column). For
example, a Fractogel S column can be equilibrated with an
equilibration buffer comprising 20 mM sodium phosphate, 25 mM
sodium chloride, the mixture can be applied to the column, the
column can be at least washed once with equilibration buffer and
the first eluate can be obtained by eluting with an elution buffer
comprising 20 mM sodium phosphate, 150 mM sodium chloride.
[0069] Preferably, the anion exchange resin is a Q sepharose column
For example, a Q sepharose column can be equilibrated with an
equilibration buffer comprising 25 mM trolamine, 40 mM sodium
chloride, pH 7.6.
[0070] Preferably, the hydrophobic interaction column is a phenyl
sepharose column. For example, a phenyl sepharose column can be
equilibrated with an equilibration buffer comprising 20 mM sodium
phosphate, 1.1 M (NH.sub.4).sub.2SO.sub.4, pH 7, the first
flowthrough can be applied to the column, the column can be at
least washed once with equilibration buffer and the second eluate
can be obtained by performing a salt step-gradient to 11 mM sodium
phosphate, 0.625 M (NH.sub.4).sub.2SO.sub.4, pH 7.0.
[0071] Preferably, pH viral inactivation is achieved by maintaining
the first eluate at pH 3.5 for approximately one hour.
[0072] In yet another aspect, the invention pertains to a method
for producing a host cell protein-(HCP) reduced adalimumab
preparation from a mixture comprising adalimumab and at least one
HCP, the method comprising:
[0073] (a) applying the mixture to a cation exchange resin, wherein
the mixture is not subjected to protein A capture prior to applying
the mixture to the first ion exchange resin, to obtain a first
eluate;
[0074] (b) subjecting the first eluate to pH viral inactivation to
obtain a virally inactivated preparation;
[0075] (c) applying the virally inactivated preparation to an anion
ion exchange resin to obtain a first flow through; and
[0076] (c) applying the first flow through to a hydrophobic
interaction column such that a second eluate is obtained;
[0077] such that the HCP-reduced adalimumab preparation is
obtained.
[0078] Preferably, the cation exchange resin is a Fractogel S
column, the anion exchange resin is a Q sepharose column and the
hydrophobic interaction column is a phenyl sepharose column. For
example, a Fractogel S column can be equilibrated with an
equilibration buffer comprising 20 mM sodium phosphate, 25 mM
sodium chloride, the mixture can be applied to the column, the
column can be at least washed once with equilibration buffer and
the first eluate can be obtained by eluting with an elution buffer
comprising 20 mM sodium phosphate, 150 mM sodium chloride. Also for
example, a Q sepharose column can be equilibrated with an
equilibration buffer comprising 25 mM trolamine, 40 mM sodium
chloride, pH 7.6. Also for example, a phenyl sepharose column can
be equilibrated with an equilibration buffer comprising 20 mM
sodium phosphate, 1.1 M (NH.sub.4).sub.2SO.sub.4, pH 7, the first
flowthrough can be applied to the column, the column can be at
least washed once with equilibration buffer and the second eluate
can be obtained by performing a salt step-gradient to 11 mM sodium
phosphate, 0.625 M (NH.sub.4).sub.2SO.sub.4, pH 7.0. Also for
example, pH viral inactivation can be achieved by maintaining the
first eluate at pH 3.5 for approximately one hour.
[0079] With respect to all of the above-described purification
methods, in a preferred embodiment of the invention, the antibody
is an anti-tumor necrosis factor-alpha (TNF.alpha.) antibody, or
antigen-binding portion thereof. In one embodiment, the
anti-TNF.alpha. antibody, or antigen-binding portion thereof, is a
chimeric antibody, a humanized antibody or a multivalent antibody.
In one embodiment, the anti-TNF.alpha. antibody, or antigen-binding
portion thereof, is infliximab or golimumab.
[0080] In another embodiment, the
anti-TNF.alpha..quadrature.antibody, or antigen-binding portion
thereof, is a human antibody. In one embodiment, the
anti-TNF.alpha..quadrature.antibody, or antigen-binding portion
thereof, is an isolated human antibody that dissociates from human
TNF.alpha. with a K.sub.d of 1.times.10.sup.-8 M or less and a
K.sub.off rate constant of 1.times.10.sup.-3 s.sup.-1 or less, both
determined by surface plasmon resonance, and neutralizes human
TNF.alpha. cytotoxicity in a standard in vitro L929 assay with an
IC.sub.50 of 1.times.10.sup.-7 M or less.
[0081] In another embodiment, the anti-TNF.alpha. antibody, or
antigen-binding portion thereof, is an isolated human antibody with
the following characteristics:
[0082] a) dissociates from human TNF.alpha. with a K.sub.off rate
constant of 1.times.10.sup.-3 s.sup.-1 or less, as determined by
surface plasmon resonance;
[0083] b) has a light chain CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single
alanine substitution at position 1, 4, 5, 7 or 8 or by one to five
conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8
and/or 9;
[0084] c) has a heavy chain CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single
alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or
by one to five conservative amino acid substitutions at positions
2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.
[0085] In still another embodiment, the anti-TNF.alpha. antibody,
or antigen-binding portion thereof, is an isolated human antibody
with a light chain variable region (LCVR) comprising the amino acid
sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR)
comprising the amino acid sequence of SEQ ID NO: 2
[0086] In yet another embodiment, the anti-TNF.alpha. antibody, or
antigen-binding portion thereof, is adalimumab.
[0087] The invention provides an antibody preparation which is
substantially free of HCP as measured by a HCP ELISA produced using
any of the methods of the invention. The invention also provides a
pharmaceutical composition comprising an HCP-reduced antibody
preparation produced using any of the methods of the invention, and
a pharmaceutically acceptable carrier.
[0088] The invention includes a pharmaceutical composition
comprising an antibody an HCP-reduced antibody, wherein the level
of HCP comprises no greater than about 70 ng of HCP per mg of
antibody as measured by a HCP ELISA, and a pharmaceutically
acceptable carrier. In one embodiment, the level of HCP comprises
no greater than about 13 ng of HCP per mg of antibody as measured
by a HCP ELISA. In another embodiment, the level of HCP comprises
no greater than about 5 ng of HCP per mg of antibody as measured by
a HCP ELISA.
[0089] The invention provides a composition comprising an antibody,
wherein said composition has no detectable level of HCP as
determined by a HCP ELISA assay.
[0090] The invention also provides an antibody preparation which is
substantially free of procathepsin L produced using any of the
methods described herein. The invention also includes a
pharmaceutical composition comprising a procathepsin L-reduced
antibody preparation produced using any of the methods described
herein, and a pharmaceutically acceptable carrier.
[0091] The invention provides a pharmaceutical composition
comprising an antibody a procathepsin L-reduced antibody and a
pharmaceutically acceptable carrier, wherein the level of
procathepsin L is no greater than a cathepsin activity of about 3.0
RFU/s/mg of antibody.
[0092] With respect to all of the above-described antibody
preparations and pharmaceutical compositions, preferably the
antibody is an anti-tumor necrosis factor-alpha (TNF.alpha.)
antibody, or antigen-binding portion thereof. In one embodiment,
the anti-TNF.alpha. antibody, or antigen-binding portion thereof,
is an antibody selected from the group consisting of humanized,
chimeric or multivalent. In one embodiment, the anti-TNF.alpha.
antibody, or antigen-binding portion thereof, is infliximab or
golimumab.
[0093] In another embodiment, the anti-TNF.alpha..quadrature.
antibody, or antigen-binding portion thereof, is a human antibody.
In one embodiment, the anti-TNF.alpha..quadrature.antibody, or
antigen-binding portion thereof, is an isolated human antibody that
dissociates from human TNF.alpha. with a K.sub.d of
1.times.10.sup.-8 M or less and a K.sub.off rate constant of
1.times.10.sup.-3 s.sup.-1 or less, both determined by surface
plasmon resonance, and neutralizes human TNF.alpha. cytotoxicity in
a standard in vitro L929 assay with an IC.sub.50 of
1.times.10.sup.-7 M or less.
[0094] In another embodiment, the anti-TNF.alpha. antibody, or
antigen-binding portion thereof, is an isolated human antibody with
the following characteristics:
[0095] a) dissociates from human TNF.alpha. with a K.sub.off rate
constant of 1.times.10.sup.-3 s.sup.-1 or less, as determined by
surface plasmon resonance;
[0096] b) has a light chain CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single
alanine substitution at position 1, 4, 5, 7 or 8 or by one to five
conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8
and/or 9;
[0097] c) has a heavy chain CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single
alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or
by one to five conservative amino acid substitutions at positions
2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.
[0098] In still another embodiment, the anti-TNF.alpha. antibody,
or antigen-binding portion thereof, is an isolated human antibody
with a light chain variable region (LCVR) comprising the amino acid
sequence of SEQ ID NO: 1 and a heavy chain variable region (HCVR)
comprising the amino acid sequence of SEQ ID NO: 2
[0099] In yet another embodiment, the anti-TNF.alpha. antibody, or
antigen-binding portion thereof, is adalimumab.
[0100] The invention includes a method of treating a disorder in
which TNF.alpha. activity is detrimental comprising administering
to a human subject a pharmaceutical compositions comprising an
antibody obtained using any of the methods of the invention. In one
embodiment, the preparation is administering to the human subject
over a prolonged period of time. In one embodiment, the prolonged
period of time includes at least about 3 months, at least about 4
months or at least about 5 months.
[0101] In one embodiment, the disorder in which TNF.alpha. activity
is detrimental is selected from the group consisting of an
autoimmune disorder, an intestinal disorder, and a skin disease. In
one embodiment, the autoimmune disorder is selected from the group
consisting of rheumatoid arthritis, rheumatoid spondylitis,
osteoarthritis, gouty arthritis, an allergy, multiple sclerosis,
psoriatic arthritis, autoimmune diabetes, autoimmune uveitis,
nephrotic syndrome, and juvenile rheumatoid arthritis. In another
embodiment, the intestinal disorder is Crohn's disease. In still
another embodiment, the skin disease is psoriasis.
[0102] In one embodiment, the pharmaceutical composition is
administering in combination with an additional therapeutic agent.
In one embodiment, the additional therapeutic agent is
methotrexate.
[0103] The invention includes a method of treating a disorder in
which TNF.alpha. activity is detrimental comprising administering
to a human subject the pharmaceutical composition comprising an
antibody obtained using any of the methods of the invention. In one
embodiment, the preparation is administering to a human subject
over a prolonged period of time. In one embodiment, the prolonged
period of time includes at least about 3 months, at least about 4
months or at least about 5 months. In one embodiment, the disorder
in which TNF.alpha. activity is detrimental is selected from the
group consisting of an autoimmune disorder, an intestinal disorder,
and a skin disease. In one embodiment, the autoimmune disorder is
selected from the group consisting of rheumatoid arthritis,
rheumatoid spondylitis, osteoarthritis, gouty arthritis, an
allergy, multiple sclerosis, psoriatic arthritis, autoimmune
diabetes, autoimmune uveitis, nephrotic syndrome and juvenile
rheumatoid arthritis. In one embodiment, the intestinal disorder is
Crohn's disease. In one embodiment, the skin disease is
psoriasis.
[0104] In one embodiment, the pharmaceutical composition is
administered in combination with an additional therapeutic agent.
In one embodiment, the additional therapeutic agent is
methotrexate.
[0105] The invention provides an article of manufacture comprising
a packaging material, adalimumab, and a label or package insert
contained within the packaging material indicating that the
adalimumab formulation comprises no greater than about 70 ng of HCP
per mg of adalimumab. In one embodiment, the about 70 ng of HCP per
mg of adalimumab is measured by a HCP ELISA.
[0106] The invention also provides an article of manufacture
comprising a packaging material, adalimumab, and a label or package
insert contained within the packaging material indicating that the
adalimumab formulation comprises no greater than about 13 ng of HCP
per mg of adalimumab. In one embodiment, the about 13 ng of HCP per
mg of adalimumab is measured by a HCP ELISA.
[0107] The invention includes an article of manufacture comprising
a packaging material, adalimumab, and a label or package insert
contained within the packaging material indicating that the
adalimumab formulation comprises no greater than about 5 ng of HCP
per mg of adalimumab. In one embodiment, the about 5 ng of HCP per
mg of adalimumab is measured by a HCP ELISA.
[0108] The invention includes an article of manufacture comprising
a packaging material, adalimumab, and a label or package insert
contained within the packaging material indicating that the
adalimumab formulation comprises no greater a level of procathepsin
L than that indicated by a cathepsin L activity of about 3.0
RFU/s/mg adalimumab. In one embodiment, cathepsin L activity is
measured by a cathepsin L kinetic assay.
[0109] The invention further provides a kinetic assay for
determining the amount of procathepsin L in a material derived from
a mammalian cell expression system comprising contacting the
material derived from a mammalian cell expression system with an
enzyme to process procathepsin L to an active cathepsin L form,
such that a cathepsin L sample is obtained; contacting the
cathepsin L sample with a substrate for cathepsin L; and
determining the cathepsin L activity in the cathepsin L sample as
an indication of the amount procathepsin L in the material derived
from the mammalian cell expression system. In one embodiment, the
mammalian cell expression system is Chinese Hamster Ovary (CHO)
cells. In another embodiment, the enzyme to process procathepsin L
is an endopeptidase. In still another embodiment, the substrate for
cathepsin L comprises a label. In still another embodiment, the
label is a fluorescent agent. In one embodiment, the fluorescent
agent comprises a fluorescent 7-amino-4-methyl coumarin (AMC)
group. In one embodiment, the substrate for cathepsin L comprises
Z-leucine-arginine. In still another embodiment, the
Z-leucine-arginine comprises an AMC group.
BRIEF DESCRIPTION OF THE DRAWINGS
[0110] FIGS. 1A and 1B show the typical elution profiles for the
Fractogel S chromatography step for each process, including the
process of the invention (FIG. 1A) and process A (FIG. 1B).
[0111] FIGS. 2A and 2B show a comparison of the flow-through wash
profile of Q Sepharose FF chromatography step, including the
process of the invention (FIG. 2A) and process A (FIG. 2B).
[0112] FIGS. 3A and 3B show a comparison of the elution profile of
Phenyl Sepharose HP chromatography step, including process B (FIG.
3A) and process A (FIG. 3B).
[0113] FIG. 4 shows a graphic depiction of a stepwise reduction in
procathepsin L for the average process B (diamond shape) and
average process A (square shape).
[0114] FIG. 5 shows a graphic depiction of the stepwise reduction
in HCP for the average process B (diamond shape) and process A
(square shape).
[0115] FIG. 6 shows that kinetic readings of activated in-process
samples indicated the linear relationship of reaction time versus
fluorescent signal.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0116] In order that the present invention may be more readily
understood, certain terms are first defined.
[0117] The term "mixture", as used herein, refers to a material
having viscosity which is to be purified comprising at least one
antibody of interest which is sought to be purified from other
substances which may also be present. Mixtures can, for example, be
aqueous solutions, organic solvent systems, or aqueous/organic
solvent mixtures or solutions. The mixtures are often complex
mixtures or solutions comprising many biological molecules (such as
proteins, antibodies, hormones, and viruses), small molecules (such
as salts, sugars, lipids, etc.) and even particulate matter. While
a typical mixture of biological origin may begin as an aqueous
solution or suspension, it may also contain organic solvents used
in earlier separation steps such as solvent precipitations,
extractions, and the like. Examples of mixtures that may contain
valuable biological substances amenable to the purification by
various embodiments the present invention include, but are not
limited to, a culture supernatant from a bioreactor, a homogenized
cell suspension, plasma, plasma fractions, and milk.
[0118] By "purifying" an antibody from a mixture comprising the
antibody and one or more substances is meant increasing the degree
of purity of the antibody in the mixture by removing (completely or
partially) at least one substance from the composition. The
substance may be an impurity or contaminant, such as, but not
limited to, a host cell protein (HCP).
[0119] The term "host cell protein(s)" or "HCP(s)" refers to
proteins in the mixture that are different from the antibody of
interest and typically originate from the source of the antibody
production. HCPs are desirably excluded from the final antibody
preparation.
[0120] The term "reduced" refers to the lessening or diminishing
the amount of a substance. A reduced preparation includes a
preparation which has less of a substance, such as HCPs or
procathepsin L, relative to an initial amount. In one embodiment,
the substance is an impurity or contaminant. In one embodiment, the
term "reduced" means substantially less of the substance. In
another embodiment, the term "reduced" means no amount of the
substance. In one embodiment, no amount of a substance includes "no
detectable amount" using assays described herein.
[0121] The term "substantially free" includes no amount of a
substance, but can also include a minimal amount of a substance. In
one embodiment, no amount of a substance includes "no detectable
amount" using assays described herein.
[0122] The term "host cell protein-(HCP-) reduced" refers to a
composition, including, but not limited to, an eluate, an
preparation, a flowthrough, comprising an antibody and a lessened
or diminished amount of HCP(s) following one or more purification
steps. In one embodiment, the term "HCP-reduced" means
substantially less of the HCP(s) in the composition comprising an
antibody. In another embodiment, the term "HCP-reduced" means no
amount of the HCP(s) in the composition comprising an antibody. In
one embodiment, the term "HCP-reduced" means no detectable amount
using assays described herein in the composition comprising an
antibody.
[0123] The term "procathepsin L-reduced" refers to a composition,
including, but not limited to, an eluate, an preparation, a
flowthrough, comprising an antibody and a lessened or diminished
amount of procathepsin L following one or more purification steps.
In one embodiment, the term "procathepsin L-reduced" means
substantially less of the HCP(s) in the composition comprising an
antibody. In another embodiment, the term "procathepsin L-reduced"
means no amount of the HCP(s) in the composition comprising an
antibody. In one embodiment, the term "procathepsin L-reduced"
means no detectable amount using assays described herein in the
composition comprising an antibody.
[0124] The term "reproducibly low" refers to an ability to
consistently achieve a lessened or diminished amount, such as an
ability to achieve a lessened or diminished amount at least 80% of
the time, more preferably at least 90% of the time, more preferably
at least 95% of the time and even more preferably at least 98% of
the time.
[0125] The term "ion exchange separation step" refers to a step
where undesired substances or impurities, e.g., HCPs or
procathepsin L, are set apart from an antibody of interest based on
differences in the ionic interactions of the antibody of interest
and the undesired substance with a charged material. An example of
an ion exchange separation step includes, but is not limited to,
ion exchange chromatography, including anion exchange
chromatography and cation exchange chromatography.
[0126] "Ion exchange material" refers to an ionic material which is
used as the basis for the separation of the undesired substances or
impurities, e.g., HCPs or procathepsin L, from the antibody.
Examples of ion exchange materials include anionic and cationic
resins.
[0127] "Cation exchange material" refers to an ion exchange resin
with covalently bound negatively charged ligands, and which thus
has free cations for exchange with cations in a solution with which
the resin is contacted. A wide variety of cation exchange resins
are known in the art, for example, those wherein the covalently
bound groups are carboxylate or sulfonate. Commercially available
cation exchange resins include CMC-cellulose, SP-Sephadex.TM., and
Fast S-Sepharose.TM. (the latter two being commercially available
from Pharmacia).
[0128] "Anion exchange material" refers to an ion exchange resin
with covalently bound positively charged groups, such as quaternary
amino groups. Commercially available anion exchange resins include
DEAE cellulose, TMAE, QAE Sephadex.TM., and Fast Q Sepharose.TM.
(the latter two being commercially available from Pharmacia).
[0129] By "binding" a molecule to an ion exchange material is meant
exposing the molecule to the ion exchange material under
appropriate conditions (pH/conductivity) such that the molecule is
reversibly immobilized in or on the ion exchange material by virtue
of ionic interactions between the molecule and a charged group or
charged groups of the ion exchange material.
[0130] The term "hydrophobic interaction step" refers to a step
where undesired substances, e.g., HCPs or procathepsin L, are set
apart from an antibody of interest based on the differences in the
hydrophobic interactions of the antibody of interest and the
undesired substance with a hydrophobic material.
[0131] The term "hydrophobic interaction material" refers to a
hydrophobic material which is used as the basis for the separation
of the undesired substances, e.g., HCPs or procathepsin L, and the
antibody. Examples of hydrophobic interaction materials include
hydrophobic ligands such as alkyl groups having from about 2 to
about 8 carbon atoms, or aryl groups such as phenyl.
[0132] The term "washing" or "wash step" includes passing an
appropriate buffer through or over a given material, e.g., ion
exchange material or hydrophobic interaction material.
[0133] The term "plurality of wash steps" includes more than one
successive wash steps The successive buffers may have varying
properties such as pH, conductivity, solvent concentration, etc.,
designed to dissociate and remove varying types of impurities that
are non-specifically associated with the given material, e.g., ion
exchange material or hydrophobic interaction material. In one
embodiment, the plurality of wash steps includes an intermediate
wash, further comprising about 40-50% elution buffer.
[0134] To "elute" a molecule (e.g. antibody or contaminant
substance) from a material is meant to remove the molecule there
from by altering the buffer surrounding the material and thereby
decreasing the interaction of the molecule and the material. In one
embodiment, an antibody is eluted from an ion exchange column
wherein the buffer competes with the antibody for the charged sites
on the ion exchange material.
[0135] The term "eluate" refers to liquid comprising the molecule,
(e.g. antibody or contaminant substance) which was obtained
subsequent to the binding of the antibody of interest to a
chromatography material and addition of an elution buffer to
dissociate the antibody. Eluates may be referred to with respect to
the step in the purification process. For example, the term "first
eluate" refers to the eluate from the first chromatographic step,
the term "second eluate" refers to the eluate from the second
chromatographic step, etc.
[0136] The term "flowthrough" refers to a liquid comprising a
molecule (e.g. antibody or contaminant substance) which was
obtained by passing a mixture comprising the molecule over a
chromatography material such that the molecule passes over the
material without binding.
[0137] A "buffer" refers to a substance which, by its presence in
solution, increases the amount of acid or alkali that must be added
to cause unit change in pH. A buffered solution resists changes in
pH by the action of its acid-base conjugate components. Buffered
solutions for use with biological reagents are generally capable of
maintaining a constant concentration of hydrogen ions such that the
pH of the solution is within a physiological range. Traditional
buffer components include, but are not limited to, organic and
inorganic salts, acids and bases. Exemplary buffers for use in
purification of biological molecules (e.g., antibodies) include the
zwitterlonic or "Good" Buffers, see e.g., Good et al. (1966)
Biochemistry 5:467 and Good and Izawa (1972) Methods Enzymol.
24:62. Exemplary buffers include but are not limited to TES, MES,
PIPES, HEPES, MOPS, MOPSO, TRICINE and BICINE.
[0138] "Wash buffer" as used herein all refer herein to the
substance used to carry away impurities from the given material,
e.g., ion exchange material or hydrophobic interaction material, to
which the antibody is bound.
[0139] The "elution buffer" refers to a substance that is used to
dissociate the antibody from the given material, e.g., ion exchange
material or hydrophobic interaction material, after it has been
washed with one or more wash substances. The elution buffer acts to
dissociate the antibody. Typical elution substances are well known
in the art and may have higher concentrations of salts, free
affinity ligands or analogs, or other substances that promote
dissociation of the target substance, e.g., antibody from the given
material. The conductivity and/or pH of the elution buffer is/are
such that the antibody is eluted from the ion exchange or
hydrophobic interaction material.
[0140] The term "conductivity" refers to the ability of an aqueous
solution to conduct an electric current between two electrodes. In
solution, the current flows by ion transport. Therefore, with an
increasing amount of ions present in the aqueous solution, the
solution will have a higher conductivity. The unit of measurement
for conductivity is mmhos (mS/cm), and can be measured using a
conductivity meter sold, e.g., by Orion. The conductivity of a
solution may be altered by changing the concentration of ions
therein. For example, the concentration of a buffering agent and/or
concentration of a salt (e.g. NaCl or KCl) in the solution may be
altered in order to achieve the desired conductivity. In one
embodiment, the salt concentration of a wash buffer or any other
aqueous solution used in chromatography is modified to achieve the
desired conductivity.
[0141] The "pI" or "isoelectric point" of a polypeptide, such as an
antibody, refers to the pH at which the polypeptide's positive
charge balances its negative charge. pI can be calculated from the
net charge of the amino acid residues of the polypeptide or can be
determined by isoelectric focusing.
[0142] The term "viral inactivation" includes rendering a virus
contained in the mixture nonfunctional or removing a virus from the
mixture to be purified. The virus may originate from the source of
antibody production, downstream processing steps or manufacturing
conditions. Methods of rendering a virus nonfunctional or removing
a virus include heat activation, pH inactivation, chemical
inactivating agents, etc. The term "pH viral inactivation" includes
subjecting a virus to a pH sufficient to render the virus
nonfunctional.
[0143] The term "human TNF.alpha." (abbreviated herein as
hTNF.alpha., or simply hTNF), as used herein, is intended to refer
to a human cytokine that exists as a 17 kD secreted form and a 26
kD membrane associated form, the biologically active form of which
is composed of a trimer of noncovalently bound 17 kD molecules. The
structure of hTNF.alpha. is described further in, for example,
Pennica, D., et al. (1984) Nature 312:724-729; Davis, J. M., et al.
(1987) Biochemistry 26:1322-1326; and Jones, E. Y., et al. (1989)
Nature 338:225-228. The term human TNF.alpha. is intended to
include recombinant human TNF.alpha. (rhTNF.alpha.), which can be
prepared by standard recombinant expression methods or purchased
commercially (R & D Systems, Catalog No. 210-TA, Minneapolis,
Minn.). TNF.alpha. is also referred to as TNF.
[0144] The term "antibody", as used herein, is intended to refer to
immunoglobulin molecules comprised of four polypeptide chains, two
heavy (H) chains and two light (L) chains inter-connected by
disulfide bonds. Each heavy chain is comprised of a heavy chain
variable region (abbreviated herein as HCVR or VH) and a heavy
chain constant region. The heavy chain constant region is comprised
of three domains, CH1, CH2 and CH3. Each light chain is comprised
of a light chain variable region (abbreviated herein as LCVR or VL)
and a light chain constant region. The light chain constant region
is comprised of one domain, CL. The VH and VL regions can be
further subdivided into regions of hypervariability, termed
complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each VH and VL is composed of three CDRs and four FRs, arranged
from amino-terminus to carboxy-terminus in the following order:
FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The antibodies of the
invention are described in further detail in U.S. Pat. Nos.
6,090,382; 6,258,562; and 6,509,015, each of which is incorporated
herein by reference in its entirety. In one embodiment, the
antibody of the invention is an anti-TNF.alpha. which interfere
with TNF.alpha. activity. Examples of anti-TNF.alpha. antibodies
include, but are not limited to, anti-TNF.alpha. human antibodies
and antibody portions described herein as well as those described
in U.S. Pat. Nos. 6,090,382; 6,258,562; 6,509,015, and in U.S.
patent application Ser. Nos. 09/801,185 and 10/302,356, each of
which is incorporated by reference herein. In one embodiment, the
TNF.alpha. inhibitor used in the invention is an anti-TNF.alpha.
antibody, or a fragment thereof, including infliximab
(Remicade.RTM., Johnson and Johnson; described in U.S. Pat. No.
5,656,272, incorporated by reference herein), CDP571 (a humanized
monoclonal anti-TNF-alpha IgG4 antibody), CDP 870 (a humanized
monoclonal anti-TNF-alpha antibody fragment), an anti-TNF dAb
(Peptech), CNTO 148 (golimumab; Medarex and Centocor), antibodies
described in WO 02/12502, and adalimumab (Humira.RTM. Abbott
Laboratories, a human anti-TNF mAb, described in U.S. Pat. No.
6,090,382 as D2E7). Additional TNF antibodies which may be used in
the invention are described in U.S. Pat. Nos. 6,593,458; 6,498,237;
6,451,983; and 6,448,380, each of which is incorporated by
reference herein. The term includes the "antibody of interest"
which is the antibody which is the target of the process of the
invention.
[0145] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., hTNF.alpha.). It has been shown that the
antigen-binding function of an antibody can be performed by
fragments of a full-length antibody. Examples of binding fragments
encompassed within the term "antigen-binding portion" of an
antibody include (i) a Fab fragment, a monovalent fragment
consisting of the VL, VH, CL and CH1 domains; (ii) a F(ab').sub.2
fragment, a bivalent fragment comprising two Fab fragments linked
by a disulfide bridge at the hinge region; (iii) a Fd fragment
consisting of the VH and CH1 domains; (iv) a Fv fragment consisting
of the VL and VH domains of a single arm of an antibody, (v) a dAb
fragment (Ward et al. (1989) Nature 341:544-546), which consists of
a VH domain; and (vi) an isolated complementarity determining
region (CDR). Furthermore, although the two domains of the Fv
fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that
enables them to be made as a single protein chain in which the VL
and VH regions pair to form monovalent molecules (known as single
chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423-426;
and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).
Such single chain antibodies are also intended to be encompassed
within the term "antigen-binding portion" of an antibody. Other
forms of single chain antibodies, such as diabodies are also
encompassed. Diabodies are bivalent, bispecific antibodies in which
VH and VL domains are expressed on a single polypeptide chain, but
using a linker that is too short to allow for pairing between the
two domains on the same chain, thereby forcing the domains to pair
with complementary domains of another chain and creating two
antigen binding sites (see e.g., Holliger et al. (1993) Proc. Natl.
Acad. Sci. USA 90:6444-6448; Poljak et al. (1994) Structure
2:1121-1123). The antibody portions of the invention are described
in further detail in U.S. Pat. Nos. 6,090,382, 6,258,562,
6,509,015, each of which is incorporated herein by reference in its
entirety.
[0146] Binding fragments are produced by recombinant DNA
techniques, or by enzymatic or chemical cleavage of intact
immunoglobulins. Binding fragments include Fab, Fab', F(ab').sub.2,
Fabc, Fv, single chains, and single-chain antibodies. Other than
"bispecific" or "bifunctional" immunoglobulins or antibodies, an
immunoglobulin or antibody is understood to have each of its
binding sites identical. A "bispecific" or "bifunctional antibody"
is an artificial hybrid antibody having two different heavy/light
chain pairs and two different binding sites. Bispecific antibodies
can be produced by a variety of methods including fusion of
hybridomas or linking of Fab' fragments. See, e.g., Songsivilai
& Lachmann, Clin. Exp. Immunol. 79:315-321 (1990); Kostelny et
al., J. Immunol. 148, 1547-1553 (1992).
[0147] A "conservative amino acid substitution", as used herein, is
one in which one amino acid residue is replaced with another amino
acid residue having a similar side chain. Families of amino acid
residues having similar side chains have been defined in the art,
including basic side chains (e.g., lysine, arginine, histidine),
acidic side chains (e.g., aspartic acid, glutamic acid), uncharged
polar side chains (e.g., glycine, asparagine, glutamine, serine,
threonine, tyrosine, cysteine), nonpolar side chains (e.g.,
alanine, valine, leucine, isoleucine, proline, phenylalanine,
methionine, tryptophan), beta-branched side chains (e.g.,
threonine, valine, isoleucine) and aromatic side chains (e.g.,
tyrosine, phenylalanine, tryptophan, histidine).
[0148] "Chimeric antibodies" refers to antibodies wherein one
portion of each of the amino acid sequences of heavy and light
chains is homologous to corresponding sequences in antibodies
derived from a particular species or belonging to a particular
class, while the remaining segment of the chains is homologous to
corresponding sequences from another species. In one embodiment,
the invention features a chimeric antibody or antigen-binding
fragment, in which the variable regions of both light and heavy
chains mimics the variable regions of antibodies derived from one
species of mammals, while the constant portions are homologous to
the sequences in antibodies derived from another species. In a
preferred embodiment of the invention, chimeric antibodies are made
by grafting CDRs from a mouse antibody onto the framework regions
of a human antibody.
[0149] "Humanized antibodies" refer to antibodies which comprise at
least one chain comprising variable region framework residues
substantially from a human antibody chain (referred to as the
acceptor immunoglobulin or antibody) and at least one
complementarity determining region (CDR) substantially from a
non-human-antibody (e.g., mouse). In addition to the grafting of
the CDRs, humanized antibodies typically undergo further
alterations in order to improve affinity and/or immunogenicity.
[0150] The term "multivalent antibody" refers to an antibody
comprising more than one antigen recognition site. For example, a
"bivalent" antibody has two antigen recognition sites, whereas a
"tetravalent" antibody has four antigen recognition sites. The
terms "monospecific", "bispecific", "trispecific", "tetraspecific",
etc. refer to the number of different antigen recognition site
specificities (as opposed to the number of antigen recognition
sites) present in a multivalent antibody. For example, a
"monospecific" antibody's antigen recognition sites all bind the
same epitope. A "bispecific" or "dual specific" antibody has at
least one antigen recognition site that binds a first epitope and
at least one antigen recognition site that binds a second epitope
that is different from the first epitope. A "multivalent
monospecific" antibody has multiple antigen recognition sites that
all bind the same epitope. A "multivalent bispecific" antibody has
multiple antigen recognition sites, some number of which bind a
first epitope and some number of which bind a second epitope that
is different from the first epitope
[0151] The term "human antibody", as used herein, is intended to
include antibodies having variable and constant regions derived
from human germline immunoglobulin sequences. The human antibodies
of the invention may include amino acid residues not encoded by
human germline immunoglobulin sequences (e.g., mutations introduced
by random or site-specific mutagenesis in vitro or by somatic
mutation in vivo), for example in the CDRs and in particular CDR3.
However, the term "human antibody", as used herein, is not intended
to include antibodies in which CDR sequences derived from the
germline of another mammalian species, such as a mouse, have been
grafted onto human framework sequences.
[0152] The term "recombinant human antibody", as used herein, is
intended to include all human antibodies that are prepared,
expressed, created or isolated by recombinant means, such as
antibodies expressed using a recombinant expression vector
transfected into a host cell (described further below), antibodies
isolated from a recombinant, combinatorial human antibody library
(described further below), antibodies isolated from an animal
(e.g., a mouse) that is transgenic for human immunoglobulin genes
(see e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287) or
antibodies prepared, expressed, created or isolated by any other
means that involves splicing of human immunoglobulin gene sequences
to other DNA sequences. Such recombinant human antibodies have
variable and constant regions derived from human germline
immunoglobulin sequences. In certain embodiments, however, such
recombinant human antibodies are subjected to in vitro mutagenesis
(or, when an animal transgenic for human Ig sequences is used, in
vivo somatic mutagenesis) and thus the amino acid sequences of the
VH and VL regions of the recombinant antibodies are sequences that,
while derived from and related to human germline VH and VL
sequences, may not naturally exist within the human antibody
germline repertoire in vivo.
[0153] Such chimeric, humanized, human, and dual specific
antibodies can be produced by recombinant DNA techniques known in
the art, for example using methods described in PCT International
Application No. PCT/US86/02269; European Patent Application No.
184,187; European Patent Application No. 171,496; European Patent
Application No. 173,494; PCT International Publication No. WO
86/01533; U.S. Pat. No. 4,816,567; European Patent Application No.
125,023; Better et al. (1988) Science 240:1041-1043; Liu et al.
(1987) Proc. Natl. Acad. Sci. USA 84:3439-3443; Liu et al. (1987)
J. Immunol. 139:3521-3526; Sun et al. (1987) Proc. Natl. Acad. Sci.
USA 84:214-218; Nishimura et al. (1987) Cancer Res. 47:999-1005;
Wood et al. (1985) Nature 314:446-449; Shaw et al. (1988) J. Natl.
Cancer Inst. 80:1553-1559); Morrison (1985) Science 229:1202-1207;
Oi et al. (1986) BioTechniques 4:214; U.S. Pat. No. 5,225,539;
Jones et al. (1986) Nature 321:552-525; Verhoeyan et al. (1988)
Science 239:1534; and Beidler et al. (1988) J. Immunol.
141:4053-4060, Queen et al., Proc. Natl. Acad. Sci. USA
86:10029-10033 (1989), U.S. Pat. No. 5,530,101, U.S. Pat. No.
5,585,089, U.S. Pat. No. 5,693,761, U.S. Pat. No. 5,693,762, Selick
et al., WO 90/07861, and Winter, U.S. Pat. No. 5,225,539.
[0154] An "isolated antibody", as used herein, is intended to refer
to an antibody that is substantially free of other antibodies
having different antigenic specificities (e.g., an isolated
antibody that specifically binds hTNF.alpha. is substantially free
of antibodies that specifically bind antigens other than
hTNF.alpha.). An isolated antibody that specifically binds
hTNF.alpha. may, however, have cross-reactivity to other antigens,
such as TNF.alpha. molecules from other species (discussed in
further detail below). Moreover, an isolated antibody may be
substantially free of other cellular material and/or chemicals.
[0155] A "neutralizing antibody", as used herein (or an "antibody
that neutralized hTNF.alpha. activity"), is intended to refer to an
antibody whose binding to hTNF.alpha. results in inhibition of the
biological activity of hTNF.alpha.. This inhibition of the
biological activity of hTNF.alpha. can be assessed by measuring one
or more indicators of hTNF.alpha. biological activity, such as
hTNF.alpha.-induced cytotoxicity (either in vitro or in vivo),
hTNF.alpha.-induced cellular activation and hTNF.alpha. binding to
hTNF.alpha. receptors. These indicators of hTNF.alpha. biological
activity can be assessed by one or more of several standard in
vitro or in vivo assays known in the art (see U.S. Pat. No.
6,090,382). Preferably, the ability of an antibody to neutralize
hTNF.alpha. activity is assessed by inhibition of
hTNF.alpha.-induced cytotoxicity of L929 cells. As an additional or
alternative parameter of hTNF.alpha. activity, the ability of an
antibody to inhibit hTNF.alpha.-induced expression of ELAM-1 on
HUVEC, as a measure of hTNF.alpha.-induced cellular activation, can
be assessed.
[0156] The term "surface plasmon resonance", as used herein, refers
to an optical phenomenon that allows for the analysis of real-time
biospecific interactions by detection of alterations in protein
concentrations within a biosensor matrix, for example using the
BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and
Piscataway, N.J.). For further descriptions, see Example 1 of U.S.
Pat. No. 6,258,562 and Jonsson et al. (1993) Ann. Biol. Clin.
51:19; Jonsson et al. (1991) Biotechniques 11:620-627; Johnsson et
al. (1995) J. Mol. Recognit. 8:125; and Johnnson et al. (1991)
Anal. Biochem. 198:268.
[0157] The term "K.sub.off", as used herein, is intended to refer
to the off rate constant for dissociation of an antibody from the
antibody/antigen complex.
[0158] The term "K.sub.d", as used herein, is intended to refer to
the dissociation constant of a particular antibody-antigen
interaction.
[0159] The term "IC.sub.50" as used herein, is intended to refer to
the concentration of the inhibitor required to inhibit the
biological endpoint of interest, e.g., neutralize cytotoxicity
activity.
[0160] The term "nucleic acid molecule", as used herein, is
intended to include DNA molecules and RNA molecules. A nucleic acid
molecule may be single-stranded or double-stranded, but preferably
is double-stranded DNA.
[0161] The term "isolated nucleic acid molecule", as used herein in
reference to nucleic acids encoding antibodies or antibody portions
(e.g., VH, VL, CDR3) that bind hTNF.alpha., is intended to refer to
a nucleic acid molecule in which the nucleotide sequences encoding
the antibody or antibody portion are free of other nucleotide
sequences encoding antibodies or antibody portions that bind
antigens other than hTNF.alpha., which other sequences may
naturally flank the nucleic acid in human genomic DNA. Thus, for
example, an isolated nucleic acid of the invention encoding a VH
region of an anti-hTNF.alpha. antibody contains no other sequences
encoding other VH regions that bind antigens other than
hTNF.alpha..
[0162] The term "vector", as used herein, is intended to refer to a
nucleic acid molecule capable of transporting another nucleic acid
to which it has been linked. One type of vector is a "plasmid",
which refers to a circular double stranded DNA loop into which
additional DNA segments may be ligated. Another type of vector is a
viral vector, wherein additional DNA segments may be ligated into
the viral genome. Certain vectors are capable of autonomous
replication in a host cell into which they are introduced (e.g.,
bacterial vectors having a bacterial origin of replication and
episomal mammalian vectors). Other vectors (e.g., non-episomal
mammalian vectors) can be integrated into the genome of a host cell
upon introduction into the host cell, and thereby are replicated
along with the host genome. Moreover, certain vectors are capable
of directing the expression of genes to which they are operatively
linked. Such vectors are referred to herein as "recombinant
expression vectors" (or simply, "expression vectors"). In general,
expression vectors of utility in recombinant DNA techniques are
often in the form of plasmids. In the present specification,
"plasmid" and "vector" may be used interchangeably as the plasmid
is the most commonly used form of vector. However, the invention is
intended to include such other forms of expression vectors, such as
viral vectors (e.g., replication defective retroviruses,
adenoviruses and adeno-associated viruses), which serve equivalent
functions.
[0163] The term "recombinant host cell" (or simply "host cell"), as
used herein, is intended to refer to a cell into which a
recombinant expression vector has been introduced. It should be
understood that such terms are intended to refer not only to the
particular subject cell but to the progeny of such a cell. Because
certain modifications may occur in succeeding generations due to
either mutation or environmental influences, such progeny may not,
in fact, be identical to the parent cell, but are still included
within the scope of the term "host cell" as used herein.
[0164] The term "kit" as used herein refers to a packaged product
or article of manufacture comprising components. The kit preferably
comprises a box or container that holds the components of the kit.
The box or container is affixed with a label or a Food and Drug
Administration approved protocol. The box or container holds
components of the invention which are preferably contained within
plastic, polyethylene, polypropylene, ethylene, or propylene
vessels. The vessels can be capped-tubes or bottles. The kit can
also include instructions for administering the TNF.alpha. antibody
of the invention. In one embodiment the kit of the invention
includes the formulation comprising the human antibody D2E7, as
described in PCT/IB03/04502 and U.S. application Ser. No.
10/222,140.
[0165] Various aspects of the invention are described in further
detail herein.
II. Antibody Production
[0166] The invention herein provides methods for purifying an
antibody from a mixture comprising the antibody and one or more
HCPs. The initial mixture is generally one resulting from the
recombinant production of the antibody. Alternatively, the initial
mixture may result from production of the antibody by peptide
synthesis (or other synthetic means) or the antibody may be
purified from a native source of the antibody.
[0167] To express the antibodies, or antibody portions of the
invention, DNAs encoding partial or full-length light and heavy
chains are inserted into expression vectors such that the genes are
operatively linked to transcriptional and translational control
sequences. In this context, the term "operatively linked" is
intended to mean that an antibody gene is ligated into a vector
such that transcriptional and translational control sequences
within the vector serve their intended function of regulating the
transcription and translation of the antibody gene. The expression
vector and expression control sequences are chosen to be compatible
with the expression host cell used. The antibody light chain gene
and the antibody heavy chain gene can be inserted into separate
vector or, more typically, both genes are inserted into the same
expression vector. The antibody genes are inserted into the
expression vector by standard methods (e.g., ligation of
complementary restriction sites on the antibody gene fragment and
vector, or blunt end ligation if no restriction sites are present).
Prior to insertion of the antibody or antibody-related light or
heavy chain sequences, the expression vector may already carry
antibody constant region sequences. For example, one approach to
converting the adalimumab or adalimumab-related VH and VL sequences
to full-length antibody genes is to insert them into expression
vectors already encoding heavy chain constant and light chain
constant regions, respectively, such that the VH segment is
operatively linked to the CH segment(s) within the vector and the
VL segment is operatively linked to the CL segment within the
vector. Additionally or alternatively, the recombinant expression
vector can encode a signal peptide that facilitates secretion of
the antibody chain from a host cell. The antibody chain gene can be
cloned into the vector such that the signal peptide is linked
in-frame to the amino terminus of the antibody chain gene. The
signal peptide can be an immunoglobulin signal peptide or a
heterologous signal peptide (i.e., a signal peptide from a
non-immunoglobulin protein).
[0168] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
The term "regulatory sequence" is intended to include promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel; Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). It will be appreciated by those skilled in the art that the
design of the expression vector, including the selection of
regulatory sequences may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV) (such as the CMV
promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40
promoter/enhancer), adenovirus, (e.g., the adenovirus major late
promoter (AdMLP)) and polyoma. For further description of viral
regulatory elements, and sequences thereof, see e.g., U.S. Pat. No.
5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and
U.S. Pat. No. 4,968,615 by Schaffner et al.
[0169] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr.sup.- host
cells with methotrexate selection/amplification) and the neo gene
(for G418 selection).
[0170] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. Although it is theoretically possible to express the
antibodies of the invention in either prokaryotic or eukaryotic
host cells, expression of antibodies in eukaryotic cells, and most
preferably mammalian host cells, is the most preferred because such
eukaryotic cells, and in particular mammalian cells, are more
likely than prokaryotic cells to assemble and secrete a properly
folded and immunologically active antibody. Prokaryotic expression
of antibody genes has been reported to be ineffective for
production of high yields of active antibody (Boss and Wood (1985)
Immunology Today 6:12-13).
[0171] Suitable host cells for cloning or expressing the DNA in the
vectors herein are the prokaryote, yeast, or higher eukaryote cells
described above. Suitable prokaryotes for this purpose include
eubacteria, such as Gram-negative or Gram-positive organisms, for
example, Enterobacteriaceae such as Escherichia, e.g., E. coli,
Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g.,
Salmonella typhimurium, Serratia, e.g., Serratia marcescans, and
Shigella, as well as Bacilli such as B. subtilis and B.
licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710
published Apr. 12, 1989), Pseudomonas such as P. aeruginosa, and
Streptomyces. One preferred E. coli cloning host is E. coli 294
(ATCC 31,446), although other strains such as E. coli B, E. coli
X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.
These examples are illustrative rather than limiting.
[0172] In addition to prokaryotes, eukaryotic microbes such as
filamentous fungi or yeast are suitable cloning or expression hosts
for polypeptide encoding vectors. Saccharomyces cerevisiae, or
common bakers yeast, is the most commonly used among lower
eukaryotic host microorganisms. However, a number of other genera,
species, and strains are commonly available and useful herein, such
as Schizosaccharomyces pombe; Kluyveromyces hosts such as, e.g., K.
lactis, K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K.
wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum
(ATCC 36,906), K. thermotolerans, and K. marxianus; yarrowia (EP
402,226); Pichia pastoris (EP 183,070); Candida; Trichoderma reesia
(EP 244,234); Neurospora crassa; Schwanniomyces such as
Schwanniomyces occidentalis; and filamentous fungi such as, e.g.,
Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such
as A. nidulans and A. niger.
[0173] Suitable host cells for the expression of glycosylated
antibodies are derived from multicellular organisms. Examples of
invertebrate cells include plant and insect cells. Numerous
baculoviral strains and variants and corresponding permissive
insect host cells from hosts such as Spodoptera frugiperda
(caterpillar), Aedes aegypti (mosquito), Aedes albopictus
(mosquito), Drosophila melanogaster (fruitfly), and Bombyx mori
have been identified. A variety of viral strains for transfection
are publicly available, e.g., the L-1 variant of Autographa
californica NPV and the Bm-5 strain of Bombyx mori NPV, and such
viruses may be used as the virus herein according to the present
invention, particularly for transfection of Spodoptera frugiperda
cells. Plant cell cultures of cotton, corn, potato, soybean,
petunia, tomato, and tobacco can also be utilized as hosts.
[0174] Preferred mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub
and ChasM, (1980) PNAS USA 77:4216-4220, used with a DHFR
selectable marker, e.g., as described in Kaufman and Sharp (1982)
Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells and SP2
cells. When recombinant expression vectors encoding antibody genes
are introduced into mammalian host cells, the antibodies are
produced by culturing the host cells for a period of time
sufficient to allow for expression of the antibody in the host
cells or, more preferably, secretion of the antibody into the
culture medium in which the host cells are grown. Other examples of
useful mammalian host cell lines are monkey kidney CV1 line
transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney
line (293 or 293 cells subcloned for growth in suspension culture,
Graham et al., J. Gen Virol. 36:59 (1977)); baby hamster kidney
cells (BHK, ATCC CCL 10); Chinese hamster ovary cells/-DHFR (CHO,
Urlaub et al., Proc. Natl. Acad. Sci. USA 77:4216 (1980)); mouse
sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251 (1980));
monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney
cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells
(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34);
buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells
(W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse
mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al.,
Annals N.Y. Acad. Sci. 383:44-68 (1982)); MRC 5 cells; FS4 cells;
and a human hepatoma line (Hep G2).
[0175] Host cells are transformed with the above-described
expression or cloning vectors for antibody production and cultured
in conventional nutrient media modified as appropriate for inducing
promoters, selecting transformants, or amplifying the genes
encoding the desired sequences.
[0176] The host cells used to produce an antibody may be cultured
in a variety of media. Commercially available media such as Ham's
F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640
(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are
suitable for culturing the host cells. In addition, any of the
media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes et
al., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704;
4,657,866; 4,927,762; 4,560,655; or 5,122,469; WO 90/03430; WO
87/00195; or U.S. Pat. No. Re. 30,985 may be used as culture media
for the host cells. Any of these media may be supplemented as
necessary with hormones and/or other growth factors (such as
insulin, transferrin, or epidermal growth factor), salts (such as
sodium chloride, calcium, magnesium, and phosphate), buffers (such
as HEPES), nucleotides (such as adenosine and thymidine),
antibiotics (such as gentamycin drug), trace elements (defined as
inorganic compounds usually present at final concentrations in the
micromolar range), and glucose or an equivalent energy source.
[0177] Any other necessary supplements may also be included at
appropriate concentrations that would be known to those skilled in
the art. The culture conditions, such as temperature, pH, and the
like, are those previously used with the host cell selected for
expression, and will be apparent to the ordinarily skilled
artisan.
[0178] Host cells can also be used to produce portions of intact
antibodies, such as Fab fragments or scFv molecules. It is
understood that variations on the above procedure are within the
scope of the present invention. For example, it may be desirable to
transfect a host cell with DNA encoding either the light chain or
the heavy chain (but not both) of an antibody of this invention.
Recombinant DNA technology may also be used to remove some or all
of the DNA encoding either or both of the light and heavy chains
that is not necessary for binding to hTNF.alpha.. The molecules
expressed from such truncated DNA molecules are also encompassed by
the antibodies of the invention. In addition, bifunctional
antibodies may be produced in which one heavy and one light chain
are an antibody of the invention and the other heavy and light
chain are specific for an antigen other than hTNF.alpha. by
crosslinking an antibody of the invention to a second antibody by
standard chemical crosslinking methods.
[0179] In a preferred system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr-CHO
cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to CMV enhancer/AdMLP promoter
regulatory elements to drive high levels of transcription of the
genes. The recombinant expression vector also carries a DHFR gene,
which allows for selection of CHO cells that have been transfected
with the vector using methotrexate selection/amplification. The
selected transformant host cells are culture to allow for
expression of the antibody heavy and light chains and intact
antibody is recovered from the culture medium. Standard molecular
biology techniques are used to prepare the recombinant expression
vector, transfect the host cells, select for transformants, culture
the host cells and recover the antibody from the culture
medium.
[0180] Recombinant human antibodies of the invention, including
adalimumab or an antigen binding portion thereof, or
adalimumab-related antibodies disclosed herein can be isolated by
screening of a recombinant combinatorial antibody library,
preferably a scFv phage display library, prepared using human VL
and VH cDNAs prepared from mRNA derived from human lymphocytes.
Methodologies for preparing and screening such libraries are known
in the art. In addition to commercially available kits for
generating phage display libraries (e.g., the Pharmacia Recombinant
Phage Antibody System, catalog no. 27-9400-01; and the Stratagene
SurfZAP.TM. phage display kit, catalog no. 240612), examples of
methods and reagents particularly amenable for use in generating
and screening antibody display libraries can be found in, for
example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT
Publication No. WO 92/18619; Dower et al. PCT Publication No. WO
91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland
et al. PCT Publication No. WO 92/15679; Breitling et al. PCT
Publication No. WO 93/01288; McCafferty et al. PCT Publication No.
WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs
et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum
Antibod Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; McCafferty et al., Nature (1990) 348:552-554;
Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J
Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628;
Gram et al. (1992) PNAS 89:3576-3580; Garrard et al. (1991)
Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res
19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.
[0181] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed cells (e.g. resulting from homogenization), is
removed, for example, by centrifugation or ultrafiltration. Where
the antibody is secreted into the medium, supernatants from such
expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit.
[0182] Prior to the process of the invention, procedures for
purification of antibodies from cell debris initially depend on the
site of expression of the antibody. Some antibodies can be caused
to be secreted directly from the cell into the surrounding growth
media; others are made intracellularly. For the latter antibodies,
the first step of a purification process involves: lysis of the
cell, which can be done by a variety of methods, including
mechanical shear, osmotic shock, or enzymatic treatments. Such
disruption releases the entire contents of the cell into the
homogenate, and in addition produces subcellular fragments that are
difficult to remove due to their small size. These are generally
removed by differential centrifugation or by filtration. Where the
antibody is secreted it not he medium, supernatants from such
expression systems are generally first concentrated using a
commercially available protein concentration filter, for example,
an Amicon or Millipore Pellicon ultrafiltration unit. Where the
antibody is secreted into the medium, the recombinant host cells
can also be separated from the cell culture medium, for example, by
tangential flow filtration. Antibodies can be further recovered
from the culture medium using the antibody purification methods of
the invention.
[0183] In one embodiment, the process of the invention includes
human antibodies, or antigen-binding portions thereof, that bind to
human TNF.alpha. with high affinity and a low off rate, and have a
high neutralizing capacity. Preferably, the human antibodies are
recombinant, neutralizing human anti-hTNF.alpha. antibodies. The
most preferred recombinant, neutralizing antibody used in the
method of the invention is referred to herein as adalimumab, also
referred to as adalimumab, Humira.RTM., and D2E7 (the amino acid
sequence of the D2E7 VL region is shown in SEQ ID NO: 1; the amino
acid sequence of the D2E7 VH region is shown in SEQ ID NO: 2). The
properties of D2E7 (adalimumab; Humira.RTM.) have been described in
Salfeld et al., U.S. Pat. Nos. 6,090,382, 6,258,562, and 6,509,015,
which are each incorporated by reference herein. Other examples of
TNF.alpha. antibodies include chimeric and humanized murine
anti-hTNF.alpha. antibodies which have undergone clinical testing
for treatment of rheumatoid arthritis (see e.g., Elliott et al.
(1994) Lancet 344:1125-1127; Elliot et al. (1994) Lancet
344:1105-1110; Rankin et al. (1995) Br. J. Rheumatol. 34:334-342).
In another embodiment, the TNF.alpha. antibody used in the
invention is infliximab (Remicade.RTM., Johnson and Johnson;
described in U.S. Pat. No. 5,656,272, incorporated by reference
herein), CDP571 (a humanized monoclonal anti-TNF-alpha IgG4
antibody), CDP 870 (a humanized monoclonal anti-TNF-alpha antibody
fragment), an anti-TNF dAb (Peptech), and CNTO 148 (golimumab;
Medarex and Centocor, see also WO 02/12502).
[0184] In one embodiment, the methods of the invention include
adalimumab antibodies and antibody portions, adalimumab-related
antibodies and antibody portions, and other human antibodies and
antibody portions with equivalent properties to adalimumab, such as
high affinity binding to hTNF.alpha. with low dissociation kinetics
and high neutralizing capacity. In one embodiment, the invention
provides treatment with an isolated human antibody, or an
antigen-binding portion thereof, that dissociates from human
TNF.alpha. with a K.sub.d of 1.times.10.sup.-8 M or less and a
K.sub.off rate constant of 1.times.10.sup.-3 s.sup.-1 or less, both
determined by surface plasmon resonance, and neutralizes human
TNF.alpha. cytotoxicity in a standard in vitro L929 assay with an
IC.sub.50 of 1.times.10.sup.-7 M or less. More preferably, the
isolated human antibody, or antigen-binding portion thereof,
dissociates from human TNF.alpha. with a K.sub.off of
5.times.10.sup.-4 s.sup.-1 or less, or even more preferably, with a
K.sub.off of 1.times.10.sup.-4 s.sup.-1 or less. More preferably,
the isolated human antibody, or antigen-binding portion thereof,
neutralizes human TNF.alpha. cytotoxicity in a standard in vitro
L929 assay with an IC.sub.50 of 1.times.10.sup.-8 M or less, even
more preferably with an IC.sub.50 of 1.times.10.sup.-9 M or less
and still more preferably with an IC.sub.50 of 1.times.10.sup.-10 M
or less. In a preferred embodiment, the antibody is an isolated
human recombinant antibody, or an antigen-binding portion
thereof.
[0185] It is well known in the art that antibody heavy and light
chain CDR3 domains play an important role in the binding
specificity/affinity of an antibody for an antigen. Accordingly, in
another aspect, the invention pertains to methods of treating
rheumatoid arthritis by administering human antibodies obtained
using the methods of the invention, wherein the antibodies have
slow dissociation kinetics for association with hTNF.alpha. and
that have light and heavy chain CDR3 domains that structurally are
identical to or related to those of adalimumab. Position 9 of the
adalimumab VL CDR3 can be occupied by Ala or Thr without
substantially affecting the K.sub.off. Accordingly, a consensus
motif for the adalimumab VL CDR3 comprises the amino acid sequence:
Q-R-Y-N-R-A-P-Y-(T/A) (SEQ ID NO: 3). Additionally, position 12 of
the adalimumab VH CDR3 can be occupied by Tyr or Asn, without
substantially affecting the K.sub.off. Accordingly, a consensus
motif for the adalimumab VH CDR3 comprises the amino acid sequence:
V-S-Y-L-S-T-A-S-S-L-D-(Y/N) (SEQ ID NO: 4). Moreover, as
demonstrated in Example 2 of U.S. Pat. No. 6,090,382, the CDR3
domain of the adalimumab heavy and light chains is amenable to
substitution with a single alanine residue (at position 1, 4, 5, 7
or 8 within the VL CDR3 or at position 2, 3, 4, 5, 6, 8, 9, or 11
within the VH CDR3) without substantially affecting the K.sub.off.
Still further, the skilled artisan will appreciate that, given the
amenability of the adalimumab VL and VH CDR3 domains to
substitutions by alanine, substitution of other amino acids within
the CDR3 domains may be possible while still retaining the low off
rate constant of the antibody, in particular substitutions with
conservative amino acids. Preferably, no more than one to five
conservative amino acid substitutions are made within the
adalimumab VL and/or VH CDR3 domains. More preferably, no more than
one to three conservative amino acid substitutions are made within
the adalimumab VL and/or VH CDR3 domains. Additionally,
conservative amino acid substitutions should not be made at amino
acid positions critical for binding to hTNF.alpha.. Positions 2 and
5 of the adalimumab VL CDR3 and positions 1 and 7 of the adalimumab
VH CDR3 appear to be critical for interaction with hTNF.alpha. and
thus, conservative amino acid substitutions preferably are not made
at these positions (although an alanine substitution at position 5
of the adalimumab VL CDR3 is acceptable, as described above) (see
U.S. Pat. No. 6,090,382).
[0186] Accordingly, in another embodiment, the antibody or
antigen-binding portion thereof preferably contains the following
characteristics:
[0187] a) dissociates from human TNF.alpha. with a K.sub.off rate
constant of 1.times.10.sup.-3 s.sup.-1 or less, as determined by
surface plasmon resonance;
[0188] b) has a light chain CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single
alanine substitution at position 1, 4, 5, 7 or 8 or by one to five
conservative amino acid substitutions at positions 1, 3, 4, 6, 7, 8
and/or 9;
[0189] c) has a heavy chain CDR3 domain comprising the amino acid
sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a single
alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11 or
by one to five conservative amino acid substitutions at positions
2, 3, 4, 5, 6, 8, 9, 10, 11 and/or 12.
[0190] More preferably, the antibody, or antigen-binding portion
thereof, dissociates from human TNF.alpha. with a K.sub.off of
5.times.10.sup.-4 s.sup.-1 or less. Even more preferably, the
antibody, or antigen-binding portion thereof, dissociates from
human TNF.alpha. with a K.sub.off of 1.times.10.sup.-4 s.sup.-1 or
less.
[0191] In yet another embodiment, the antibody or antigen-binding
portion thereof preferably contains a light chain variable region
(LCVR) having a CDR3 domain comprising the amino acid sequence of
SEQ ID NO: 3, or modified from SEQ ID NO: 3 by a single alanine
substitution at position 1, 4, 5, 7 or 8, and with a heavy chain
variable region (HCVR) having a CDR3 domain comprising the amino
acid sequence of SEQ ID NO: 4, or modified from SEQ ID NO: 4 by a
single alanine substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or
11. Preferably, the LCVR further has a CDR2 domain comprising the
amino acid sequence of SEQ ID NO: 5 (i.e., the adalimumab VL CDR2)
and the HCVR further has a CDR2 domain comprising the amino acid
sequence of SEQ ID NO: 6 (i.e., the adalimumab VH CDR2). Even more
preferably, the LCVR further has CDR1 domain comprising the amino
acid sequence of SEQ ID NO: 7 (i.e., the adalimumab VL CDR1) and
the HCVR has a CDR1 domain comprising the amino acid sequence of
SEQ ID NO: 8 (i.e., the adalimumab VH CDR1). The framework regions
for VL preferably are from the V.sub..kappa.I human germline
family, more preferably from the A20 human germline Vk gene and
most preferably from the adalimumab VL framework sequences shown in
FIGS. 1A and 1B of U.S. Pat. No. 6,090,382. The framework regions
for VH preferably are from the V.sub.H3 human germline family, more
preferably from the DP-31 human germline VH gene and most
preferably from the adalimumab VH framework sequences shown in
FIGS. 2A and 2B of U.S. Pat. No. 6,090,382.
[0192] Accordingly, in another embodiment, the antibody or
antigen-binding portion thereof preferably contains a light chain
variable region (LCVR) comprising the amino acid sequence of SEQ ID
NO: 1 (i.e., the adalimumab VL) and a heavy chain variable region
(HCVR) comprising the amino acid sequence of SEQ ID NO: 2 (i.e.,
the adalimumab VH). In certain embodiments, the antibody comprises
a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4,
IgA, IgE, IgM or IgD constant region. Preferably, the heavy chain
constant region is an IgG1 heavy chain constant region or an IgG4
heavy chain constant region. Furthermore, the antibody can comprise
a light chain constant region, either a kappa light chain constant
region or a lambda light chain constant region. Preferably, the
antibody comprises a kappa light chain constant region.
Alternatively, the antibody portion can be, for example, a Fab
fragment or a single chain Fv fragment.
[0193] In still other embodiments, the antibody or antigen-binding
portion thereof preferably contains adalimumab-related VL and VH
CDR3 domains, for example, antibodies, or antigen-binding portions
thereof, with a light chain variable region (LCVR) having a CDR3
domain comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 3, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID
NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17,
SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID
NO: 22, SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25 and SEQ ID NO:
26 or with a heavy chain variable region (HCVR) having a CDR3
domain comprising an amino acid sequence selected from the group
consisting of SEQ ID NO: 4, SEQ ID NO: 27, SEQ ID NO: 28, SEQ ID
NO: 29, SEQ ID NO: 30, SEQ ID NO: 31, SEQ ID NO: 32, SEQ ID NO: 33,
SEQ ID NO: 34 and SEQ ID NO: 35.
[0194] The TNF.alpha. antibody used in the invention can be
modified. In some embodiments, the TNF.alpha. antibody or antigen
binding fragments thereof, is chemically modified to provide a
desired effect. For example, pegylation of antibodies and antibody
fragments of the invention may be carried out by any of the
pegylation reactions known in the art, as described, for example,
in the following references: Focus on Growth Factors 3:4-10 (1992);
EP 0 154 316; and EP 0 401 384 (each of which is incorporated by
reference herein in its entirety). Preferably, the pegylation is
carried out via an acylation reaction or an alkylation reaction
with a reactive polyethylene glycol molecule (or an analogous
reactive water-soluble polymer). A preferred water-soluble polymer
for pegylation of the antibodies and antibody fragments of the
invention is polyethylene glycol (PEG). As used herein,
"polyethylene glycol" is meant to encompass any of the forms of PEG
that have been used to derivatize other proteins, such as mono
(Cl--ClO) alkoxy- or aryloxy-polyethylene glycol.
[0195] Methods for preparing pegylated antibodies and antibody
fragments of the invention will generally comprise the steps of (a)
reacting the antibody or antibody fragment with polyethylene
glycol, such as a reactive ester or aldehyde derivative of PEG,
under conditions whereby the antibody or antibody fragment becomes
attached to one or more PEG groups, and (b) obtaining the reaction
products. It will be apparent to one of ordinary skill in the art
to select the optimal reaction conditions or the acylation
reactions based on known parameters and the desired result.
[0196] Pegylated antibodies and antibody fragments may generally be
used to treat TNF.alpha.-related disorders of the invention by
administration of the TNF.alpha. antibodies and antibody fragments
described herein. Generally the pegylated antibodies and antibody
fragments have increased half-life, as compared to the nonpegylated
antibodies and antibody fragments. The pegylated antibodies and
antibody fragments may be employed alone, together, or in
combination with other pharmaceutical compositions.
[0197] In yet another embodiment of the invention, TNF.alpha.
antibodies or fragments thereof can be altered wherein the constant
region of the antibody is modified to reduce at least one constant
region-mediated biological effector function relative to an
unmodified antibody. To modify an antibody of the invention such
that it exhibits reduced binding to the Fc receptor, the
immunoglobulin constant region segment of the antibody can be
mutated at particular regions necessary for Fc receptor (FcR)
interactions (see e.g., Canfield and Morrison (1991) J. Exp. Med.
173:1483-1491; and Lund et al. (1991) J. of Immunol.
147:2657-2662). Reduction in FcR binding ability of the antibody
may also reduce other effector functions which rely on FcR
interactions, such as opsonization and phagocytosis and
antigen-dependent cellular cytotoxicity.
[0198] An antibody or antibody portion of the invention can be
derivatized or linked to another functional molecule (e.g., another
peptide or protein). Accordingly, the antibodies and antibody
portions of the invention are intended to include derivatized and
otherwise modified forms of the human anti-hTNF.alpha. antibodies
described herein, including immunoadhesion molecules. For example,
an antibody or antibody portion of the invention can be
functionally linked (by chemical coupling, genetic fusion,
noncovalent association or otherwise) to one or more other
molecular entities, such as another antibody (e.g., a bispecific
antibody or a diabody), a detectable agent, a cytotoxic agent, a
pharmaceutical agent, and/or a protein or peptide that can mediate
associate of the antibody or antibody portion with another molecule
(such as a streptavidin core region or a polyhistidine tag).
[0199] One type of derivatized antibody is produced by crosslinking
two or more antibodies (of the same type or of different types,
e.g., to create bispecific antibodies). Suitable crosslinkers
include those that are heterobifunctional, having two distinctly
reactive groups separated by an appropriate spacer (e.g.,
m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional
(e.g., disuccinimidyl suberate). Such linkers are available from
Pierce Chemical Company, Rockford, Ill.
[0200] Useful detectable agents with which an antibody or antibody
portion of the invention may be derivatized include fluorescent
compounds. Exemplary fluorescent detectable agents include
fluorescein, fluorescein isothiocyanate, rhodamine,
5-dimethylamine-1-napthalenesulfonyl chloride, phycoerythrin and
the like. An antibody may also be derivatized with detectable
enzymes, such as alkaline phosphatase, horseradish peroxidase,
glucose oxidase and the like. When an antibody is derivatized with
a detectable enzyme, it is detected by adding additional reagents
that the enzyme uses to produce a detectable reaction product. For
example, when the detectable agent horseradish peroxidase is
present, the addition of hydrogen peroxide and diaminobenzidine
leads to a colored reaction product, which is detectable. An
antibody may also be derivatized with biotin, and detected through
indirect measurement of avidin or streptavidin binding.
[0201] An antibody, or antibody portion, of the invention can be
prepared by recombinant expression of immunoglobulin light and
heavy chain genes in a host cell. To express an antibody
recombinantly, a host cell is transfected with one or more
recombinant expression vectors carrying DNA fragments encoding the
immunoglobulin light and heavy chains of the antibody such that the
light and heavy chains are expressed in the host cell and,
preferably, secreted into the medium in which the host cells are
cultured, from which medium the antibodies can be recovered.
Standard recombinant DNA methodologies are used to obtain antibody
heavy and light chain genes, incorporate these genes into
recombinant expression vectors and introduce the vectors into host
cells, such as those described in Sambrook, Fritsch and Maniatis
(eds), Molecular Cloning; A Laboratory Manual, Second Edition, Cold
Spring Harbor, N.Y., (1989), Ausubel et al. (eds.) Current
Protocols in Molecular Biology, Greene Publishing Associates,
(1989) and in U.S. Pat. No. 4,816,397 by Boss et al.
[0202] To express adalimumab or a adalimumab-related antibody, DNA
fragments encoding the light and heavy chain variable regions are
first obtained. These DNAs can be obtained by amplification and
modification of germline light and heavy chain variable sequences
using the polymerase chain reaction (PCR). Germline DNA sequences
for human heavy and light chain variable region genes are known in
the art (see e.g., the "Vbase" human germline sequence database;
see also Kabat et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242; Tomlinson et al. (1992) "The
Repertoire of Human Germline V.sub.H Sequences Reveals about Fifty
Groups of V.sub.H Segments with Different Hypervariable Loops" J.
Mol. Biol. 227:776-798; and Cox et al. (1994) "A Directory of Human
Germ-line V.sub.78 Segments Reveals a Strong Bias in their Usage"
Eur. J. Immunol. 24:827-836; the contents of each of which are
expressly incorporated herein by reference). To obtain a DNA
fragment encoding the heavy chain variable region of adalimumab, or
a adalimumab-related antibody, a member of the V.sub.H3 family of
human germline VH genes is amplified by standard PCR. Most
preferably, the DP-31 VH germline sequence is amplified. To obtain
a DNA fragment encoding the light chain variable region of
adalimumab, or a adalimumab-related antibody, a member of the
V.sub..kappa.I family of human germline VL genes is amplified by
standard PCR. Most preferably, the A20 VL germline sequence is
amplified. PCR primers suitable for use in amplifying the DP-31
germline VH and A20 germline VL sequences can be designed based on
the nucleotide sequences disclosed in the references cited supra,
using standard methods.
[0203] Once the germline VH and VL fragments are obtained, these
sequences can be mutated to encode the adalimumab or
adalimumab-related amino acid sequences disclosed herein. The amino
acid sequences encoded by the germline VH and VL DNA sequences are
first compared to the adalimumab or adalimumab-related VH and VL
amino acid sequences to identify amino acid residues in the
adalimumab or adalimumab-related sequence that differ from
germline. Then, the appropriate nucleotides of the germline DNA
sequences are mutated such that the mutated germline sequence
encodes the adalimumab or adalimumab-related amino acid sequence,
using the genetic code to determine which nucleotide changes should
be made. Mutagenesis of the germline sequences is carried out by
standard methods, such as PCR-mediated mutagenesis (in which the
mutated nucleotides are incorporated into the PCR primers such that
the PCR product contains the mutations) or site-directed
mutagenesis.
[0204] Once DNA fragments encoding adalimumab or adalimumab-related
VH and VL segments are obtained (by amplification and mutagenesis
of germline VH and VL genes, as described above), these DNA
fragments can be further manipulated by standard recombinant DNA
techniques, for example to convert the variable region genes to
full-length antibody chain genes, to Fab fragment genes or to a
scFv gene. In these manipulations, a VL- or VH-encoding DNA
fragment is operatively linked to another DNA fragment encoding
another protein, such as an antibody constant region or a flexible
linker. The term "operatively linked", as used in this context, is
intended to mean that the two DNA fragments are joined such that
the amino acid sequences encoded by the two DNA fragments remain
in-frame.
[0205] The isolated DNA encoding the VH region can be converted to
a full-length heavy chain gene by operatively linking the
VH-encoding DNA to another DNA molecule encoding heavy chain
constant regions (CH1, CH2 and CH3). The sequences of human heavy
chain constant region genes are known in the art (see e.g., Kabat
et al. (1991) Sequences of Proteins of Immunological Interest,
Fifth Edition, U.S. Department of Health and Human Services, NIH
Publication No. 91-3242) and DNA fragments encompassing these
regions can be obtained by standard PCR amplification. The heavy
chain constant region can be an IgG1, IgG2, IgG3, IgG4, IgA, IgE,
IgM or IgD constant region, but most preferably is an IgG1 or IgG4
constant region. For a Fab fragment heavy chain gene, the
VH-encoding DNA can be operatively linked to another DNA molecule
encoding only the heavy chain CH1 constant region.
[0206] The isolated DNA encoding the VL region can be converted to
a full-length light chain gene (as well as a Fab light chain gene)
by operatively linking the VL-encoding DNA to another DNA molecule
encoding the light chain constant region, CL. The sequences of
human light chain constant region genes are known in the art (see
e.g., Kabat et al. (1991) Sequences of Proteins of Immunological
Interest, Fifth Edition, U.S. Department of Health and Human
Services, NIH Publication No. 91-3242) and DNA fragments
encompassing these regions can be obtained by standard PCR
amplification. The light chain constant region can be a kappa or
lambda constant region, but most preferably is a kappa constant
region.
[0207] To create a scFv gene, the VH- and VL-encoding DNA fragments
are operatively linked to another fragment encoding a flexible
linker, e.g., encoding the amino acid sequence
(Gly.sub.4-Ser).sub.3, such that the VH and VL sequences can be
expressed as a contiguous single-chain protein, with the VL and VH
regions joined by the flexible linker (see e.g., Bird et al. (1988)
Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci.
USA 85:5879-5883; McCafferty et al., Nature (1990)
348:552-554).
[0208] To express the antibodies, or antibody portions of the
invention, DNAs encoding partial or full-length light and heavy
chains, obtained as described above, are inserted into expression
vectors such that the genes are operatively linked to
transcriptional and translational control sequences. In this
context, the term "operatively linked" is intended to mean that an
antibody gene is ligated into a vector such that transcriptional
and translational control sequences within the vector serve their
intended function of regulating the transcription and translation
of the antibody gene. The expression vector and expression control
sequences are chosen to be compatible with the expression host cell
used. The antibody light chain gene and the antibody heavy chain
gene can be inserted into separate vector or, more typically, both
genes are inserted into the same expression vector. The antibody
genes are inserted into the expression vector by standard methods
(e.g., ligation of complementary restriction sites on the antibody
gene fragment and vector, or blunt end ligation if no restriction
sites are present). Prior to insertion of the adalimumab or
adalimumab-related light or heavy chain sequences, the expression
vector may already carry antibody constant region sequences. For
example, one approach to converting the adalimumab or
adalimumab-related VH and VL sequences to full-length antibody
genes is to insert them into expression vectors already encoding
heavy chain constant and light chain constant regions,
respectively, such that the VH segment is operatively linked to the
CH segment(s) within the vector and the VL segment is operatively
linked to the CL segment within the vector. Additionally or
alternatively, the recombinant expression vector can encode a
signal peptide that facilitates secretion of the antibody chain
from a host cell. The antibody chain gene can be cloned into the
vector such that the signal peptide is linked in-frame to the amino
terminus of the antibody chain gene. The signal peptide can be an
immunoglobulin signal peptide or a heterologous signal peptide
(i.e., a signal peptide from a non-immunoglobulin protein).
[0209] In addition to the antibody chain genes, the recombinant
expression vectors of the invention carry regulatory sequences that
control the expression of the antibody chain genes in a host cell.
The term "regulatory sequence" is intended to include promoters,
enhancers and other expression control elements (e.g.,
polyadenylation signals) that control the transcription or
translation of the antibody chain genes. Such regulatory sequences
are described, for example, in Goeddel; Gene Expression Technology:
Methods in Enzymology 185, Academic Press, San Diego, Calif.
(1990). It will be appreciated by those skilled in the art that the
design of the expression vector, including the selection of
regulatory sequences may depend on such factors as the choice of
the host cell to be transformed, the level of expression of protein
desired, etc. Preferred regulatory sequences for mammalian host
cell expression include viral elements that direct high levels of
protein expression in mammalian cells, such as promoters and/or
enhancers derived from cytomegalovirus (CMV) (such as the CMV
promoter/enhancer), Simian Virus 40 (SV40) (such as the SV40
promoter/enhancer), adenovirus, (e.g., the adenovirus major late
promoter (AdMLP)) and polyoma. For further description of viral
regulatory elements, and sequences thereof, see e.g., U.S. Pat. No.
5,168,062 by Stinski, U.S. Pat. No. 4,510,245 by Bell et al. and
U.S. Pat. No. 4,968,615 by Schaffner et al.
[0210] In addition to the antibody chain genes and regulatory
sequences, the recombinant expression vectors of the invention may
carry additional sequences, such as sequences that regulate
replication of the vector in host cells (e.g., origins of
replication) and selectable marker genes. The selectable marker
gene facilitates selection of host cells into which the vector has
been introduced (see e.g., U.S. Pat. Nos. 4,399,216, 4,634,665 and
5,179,017, all by Axel et al.). For example, typically the
selectable marker gene confers resistance to drugs, such as G418,
hygromycin or methotrexate, on a host cell into which the vector
has been introduced. Preferred selectable marker genes include the
dihydrofolate reductase (DHFR) gene (for use in dhfr.sup.- host
cells with methotrexate selection/amplification) and the neo gene
(for G418 selection).
[0211] For expression of the light and heavy chains, the expression
vector(s) encoding the heavy and light chains is transfected into a
host cell by standard techniques. The various forms of the term
"transfection" are intended to encompass a wide variety of
techniques commonly used for the introduction of exogenous DNA into
a prokaryotic or eukaryotic host cell, e.g., electroporation,
calcium-phosphate precipitation, DEAE-dextran transfection and the
like. Although it is theoretically possible to express the
antibodies of the invention in either prokaryotic or eukaryotic
host cells, expression of antibodies in eukaryotic cells, and most
preferably mammalian host cells, is the most preferred because such
eukaryotic cells, and in particular mammalian cells, are more
likely than prokaryotic cells to assemble and secrete a properly
folded and immunologically active antibody. Prokaryotic expression
of antibody genes has been reported to be ineffective for
production of high yields of active antibody (Boss and Wood (1985)
Immunology Today 6:12-13).
[0212] Preferred mammalian host cells for expressing the
recombinant antibodies of the invention include Chinese Hamster
Ovary (CHO cells) (including dhfr-CHO cells, described in Urlaub
and ChasM, (1980) PNAS USA 77:4216-4220, used with a DHFR
selectable marker, e.g., as described in Kaufman and Sharp (1982)
Mol. Biol. 159:601-621), NS0 myeloma cells, COS cells and SP2
cells. When recombinant expression vectors encoding antibody genes
are introduced into mammalian host cells, the antibodies are
produced by culturing the host cells for a period of time
sufficient to allow for expression of the antibody in the host
cells or, more preferably, secretion of the antibody into the
culture medium in which the host cells are grown. Antibodies can be
recovered from the culture medium using protein purification
methods.
[0213] Host cells can also be used to produce portions of intact
antibodies, such as Fab fragments or scFv molecules. It is
understood that variations on the above procedure are within the
scope of the present invention. For example, it may be desirable to
transfect a host cell with DNA encoding either the light chain or
the heavy chain (but not both) of an antibody of this invention.
Recombinant DNA technology may also be used to remove some or all
of the DNA encoding either or both of the light and heavy chains
that is not necessary for binding to hTNF.alpha.. The molecules
expressed from such truncated DNA molecules are also encompassed by
the antibodies of the invention. In addition, bifunctional
antibodies may be produced in which one heavy and one light chain
are an antibody of the invention and the other heavy and light
chain are specific for an antigen other than hTNF.alpha. by
crosslinking an antibody of the invention to a second antibody by
standard chemical crosslinking methods.
[0214] In a preferred system for recombinant expression of an
antibody, or antigen-binding portion thereof, of the invention, a
recombinant expression vector encoding both the antibody heavy
chain and the antibody light chain is introduced into dhfr-CHO
cells by calcium phosphate-mediated transfection. Within the
recombinant expression vector, the antibody heavy and light chain
genes are each operatively linked to CMV enhancer/AdMLP promoter
regulatory elements to drive high levels of transcription of the
genes. The recombinant expression vector also carries a DHFR gene,
which allows for selection of CHO cells that have been transfected
with the vector using methotrexate selection/amplification. The
selected transformant host cells are culture to allow for
expression of the antibody heavy and light chains and intact
antibody is recovered from the culture medium. Standard molecular
biology techniques are used to prepare the recombinant expression
vector, transfect the host cells, select for transformants, culture
the host cells and recover the antibody from the culture
medium.
[0215] Recombinant human antibodies of the invention in addition to
adalimumab or an antigen binding portion thereof, or
adalimumab-related antibodies disclosed herein can be isolated by
screening of a recombinant combinatorial antibody library,
preferably a scFv phage display library, prepared using human VL
and VH cDNAs prepared from mRNA derived from human lymphocytes.
Methodologies for preparing and screening such libraries are known
in the art. In addition to commercially available kits for
generating phage display libraries (e.g., the Pharmacia Recombinant
Phage Antibody System, catalog no. 27-9400-01; and the Stratagene
SurfZAP.TM. phage display kit, catalog no. 240612), examples of
methods and reagents particularly amenable for use in generating
and screening antibody display libraries can be found in, for
example, Ladner et al. U.S. Pat. No. 5,223,409; Kang et al. PCT
Publication No. WO 92/18619; Dower et al. PCT Publication No. WO
91/17271; Winter et al. PCT Publication No. WO 92/20791; Markland
et al. PCT Publication No. WO 92/15679; Breitling et al. PCT
Publication No. WO 93/01288; McCafferty et al. PCT Publication No.
WO 92/01047; Garrard et al. PCT Publication No. WO 92/09690; Fuchs
et al. (1991) Bio/Technology 9:1370-1372; Hay et al. (1992) Hum
Antibod Hybridomas 3:81-85; Huse et al. (1989) Science
246:1275-1281; McCafferty et al., Nature (1990) 348:552-554;
Griffiths et al. (1993) EMBO J. 12:725-734; Hawkins et al. (1992) J
Mol Biol 226:889-896; Clackson et al. (1991) Nature 352:624-628;
Gram et al. (1992) PNAS 89:3576-3580; Garrard et al. (1991)
Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res
19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982.
[0216] In a preferred embodiment, to isolate human antibodies with
high affinity and a low off rate constant for hTNF.alpha., a murine
anti-hTNF.alpha. antibody having high affinity and a low off rate
constant for hTNF.alpha. (e.g., MAK 195, the hybridoma for which
has deposit number ECACC 87 050801) is first used to select human
heavy and light chain sequences having similar binding activity
toward hTNF.alpha., using the epitope imprinting methods described
in Hoogenboom et al., PCT Publication No. WO 93/06213. The antibody
libraries used in this method are preferably scFv libraries
prepared and screened as described in McCafferty et al., PCT
Publication No. WO 92/01047, McCafferty et al. Nature (1990)
348:552-554; and Griffiths et al. (1993) EMBO J. 12:725-734. The
scFv antibody libraries preferably are screened using recombinant
human TNF.alpha. as the antigen.
[0217] Once initial human VL and VH segments are selected, "mix and
match" experiments, in which different pairs of the initially
selected VL and VH segments are screened for hTNF.alpha. binding,
are performed to select preferred VL/VH pair combinations.
Additionally, to further improve the affinity and/or lower the off
rate constant for hTNF.alpha. binding, the VL and VH segments of
the preferred VL/VH pair(s) can be randomly mutated, preferably
within the CDR3 region of VH and/or VL, in a process analogous to
the in vivo somatic mutation process responsible for affinity
maturation of antibodies during a natural immune response. This in
vitro affinity maturation can be accomplished by amplifying VH and
VL regions using PCR primers complimentary to the VH CDR3 or VL
CDR3, respectively, which primers have been "spiked" with a random
mixture of the four nucleotide bases at certain positions such that
the resultant PCR products encode VH and VL segments into which
random mutations have been introduced into the VH and/or VL CDR3
regions. These randomly mutated VH and VL segments can be
rescreened for binding to hTNF.alpha. and sequences that exhibit
high affinity and a low off rate for hTNF.alpha. binding can be
selected.
[0218] Following screening and isolation of an anti-hTNF.alpha.
antibody of the invention from a recombinant immunoglobulin display
library, nucleic acid encoding the selected antibody can be
recovered from the display package (e.g., from the phage genome)
and subcloned into other expression vectors by standard recombinant
DNA techniques. If desired, the nucleic acid can be further
manipulated to create other antibody forms of the invention (e.g.,
linked to nucleic acid encoding additional immunoglobulin domains,
such as additional constant regions). To express a recombinant
human antibody isolated by screening of a combinatorial library,
the DNA encoding the antibody is cloned into a recombinant
expression vector and introduced into a mammalian host cells, as
described in further detail in above.
[0219] Methods of isolating human antibodies with high affinity and
a low off rate constant for hTNF.alpha. are also described in U.S.
Pat. Nos. 6,090,382, 6,258,562, and 6,509,015, each of which is
incorporated by reference herein.
III. Antibody Purification
[0220] The invention provides an method for producing an
HCP-reduced antibody preparation from a mixture comprising an
antibody and at least one HCP. The invention also provides a method
for producing a procathepsin L-reduced antibody preparation from a
mixture comprising an antibody and at least one procathepsin L. The
purification process of the invention begins at the separation step
when the antibody has been produced using methods described in
Section II and conventional methods in the art. Typically in the
art, antibody-HCP mixtures are subjected to protein A capture
(e.g., a protein A column) as an initial purification step, since
the antibody binds to protein A whereas HCP will flow through. The
purification methods of the invention have the advantage that it is
not necessary to subject the mixture comprising an antibody and at
least one HCP to protein A capture (e.g., a protein A column) as an
initial step, or as any step in the purification method.
[0221] Once a clarified solution or mixture comprising the antibody
has been obtained, separation of the antibody from the other
proteins produced by the cell, such as HCPs, is performed using a
combination of different purification techniques, including ion
exchange separation step(s) and hydrophobic interaction separation
step(s). The separation steps separate mixtures of proteins on the
basis of their charge, degree of hydrophobicity, and/or size. In
one embodiment of the invention, separation is performed using
chromatography, including cationic, anionic, and hydrophobic.
Several different chromatography resins are available for each of
these techniques, allowing accurate tailoring of the purification
scheme to the particular protein involved. The essence of each of
the separation methods is that proteins can be caused either to
move at different rates down a long column, achieving a physical
separation that increases as they pass further down the column, or
to adhere selectively to the separation medium, being then
differentially eluted by different solvents. In some cases, the
antibody is separated from impurities when the impurities
specifically adhere to the column, and the antibody does not, that
is, the antibody is present in the flowthrough.
[0222] Methods of purifying antibodies from undesired proteins are
provided below, e.g., process A. In one embodiment, the invention
includes the steps, individually or in combination, described below
in Process A. Process A provides a method of purifying a mixture
comprising an antibody using ion exchange separation (cation
exchange chromatography and anion exchange chromatography) and
hydrophobic interaction separation, resulting in an antibody
preparation suitable for use in a pharmaceutical composition.
Process A has the advantage that it can be carried out without the
need to perform protein A capture as an initial step in antibody
purification. In one embodiment, the antibody purified using
process A is adalimumab. Process A generally comprises the
following:
[0223] A mixture comprising an antibody and impurities, e.g.,
HCP(s), is loaded onto an ion exchange column, such as a cation
exchange column. The mixture may be loaded at a load of about
.ltoreq.30 g antibody/L per cycle. The mixture loaded onto the
cation column is subsequently washed with wash buffer
(equilibration buffer). The antibody is then eluted from the
column, and a first eluate is obtained.
[0224] The first eluate is then often virally inactivated and pH
adjusted in preparation for anion exchange chromatography. The
first eluate is virally inactivated by adjusting the pH to a low pH
relative to the elution buffer (described further in section IIIC).
The pH of the virally inactivated eluate is subsequently adjusted
in more than one step to a final pH of about 7.6, which is the pH
of the anion exchange column which follows in sequence.
[0225] Following viral inactivation, the first eluate is often
subjected to a second ion exchange separation step, where the first
eluate is loaded onto an anion exchange column (e.g., a Q Sepharose
column). The column is washed with a wash buffer, and a first
flowthrough comprising the antibody is obtained.
[0226] The flowthrough is further purified by loading it onto a
hydrophobic interaction column (phenyl sepharose). The column is
washed, and the antibody is eluted from the column such that a
second eluate is obtained.
[0227] Process B is described below and provides an improved method
for producing a host cell protein-(HCP) reduced antibody
preparation from a mixture comprising an antibody. The language
"reduced" when referring to HCP or procathepsin L, includes
improvements over levels, e.g., concentration or activity, of HCP
or procathepsin L at comparable points in process A. In one
embodiment, the first eluate of process B comprises a reduced level
of HCP or procathepsin L in comparison to the first eluate of
process A. In one embodiment, the first flowthrough of process B
comprises a reduced level of HCP or procathepsin L in comparison to
the first flowthrough of process A. In one embodiment, the second
eluate of process B comprises a reduced level of HCP or
procathepsin L in comparison to the second eluate of process A. In
another embodiment, the antibody preparation resulting from process
B comprises a reduced level of HCP or procathepsin L in comparison
the antibody preparation resulting from process A.
[0228] Process B generally comprises the following:
[0229] A mixture comprising an antibody and impurities, e.g.,
HCP(s), is loaded onto an ion exchange column, such as a cation
exchange column The mixture may be loaded at a load of about
.ltoreq.35 g antibody/L per cycle at pH 7 or at a load of about
.ltoreq.70 g antibody/L per cycle at pH 5. The mixture loaded onto
the cation column is subsequently washed with wash buffer
(equilibration buffer). Following the equilibration wash buffer, an
intermediate wash step is performed, wherein the column is washed
with an intermediate buffer which has similar conductivity to the
elution buffer. This intermediate wash step improves clearance of
process-related impurities. The antibody is then eluted from the
column using elution buffer, and a first eluate is obtained.
[0230] The first eluate is then virally inactivated and pH adjusted
in preparation for anion exchange chromatography. The first eluate
is virally inactivated by adjusting the pH to a low pH relative to
the elution buffer. The pH and conductivity of the virally
inactivated eluate is subsequently adjusted in one step to a final
pH of about 7.8-8.2, which is the pH of the equilibrated anion
exchange column which follows in sequence.
[0231] Following viral inactivation, the first eluate is subjected
to a second ion exchange separation step, where the first eluate is
loaded onto an anion exchange column (e.g., a Q Sepharose column).
The column is washed with a wash buffer, and a first flowthrough
comprising the antibody is obtained.
[0232] The flowthrough is further purified by loading it onto a
hydrophobic interaction column (phenyl sepharose). The column is
washed, and the antibody is eluted from the column such that a
second eluate is obtained. The result of process B is a preparation
having reduced HCPs, including procathepsin L. Further results of
process B include the removal of process bottlenecks, e.g., higher
productivity created in cell culture scale-up, by moving HCP
clearance to the front part of the process and an overall
improvement in antibody yield. Additional details regarding the
improved process of the invention are provided below.
III.A. Ion Exchange Separation
[0233] The present invention features methods for producing a
HCP-reduced antibody preparation from a mixture comprising an
antibody and at least one HCP by subjecting the mixture to at least
one ion exchange separation step such that a first eluate
comprising the antibody is obtained. Ion exchange separation
includes any method by which two substances are separated based on
the difference in their respective ionic charges.
[0234] In performing the separation, the antibody mixture may be
contacted with the ion exchange material, e.g., using a batch
purification technique or chromatography. For example, for batch
purification, ion exchange material is prepared in or equilibrated
to the desired starting buffer. A slurry of the ion exchange
material is obtained. The antibody solution is contacted with the
slurry to adsorb the antibody to be separated to the ion exchange
material. The solution comprising the HCP(s) that do not bind to
the ion exchange material is separated from the slurry, e.g., by
allowing the slurry to settle and removing the supernatant. The
slurry can be subjected to one or more wash steps. If desired, the
slurry can be contacted with a solution of higher conductivity to
desorb HCPs that have bound to the ion exchange material. In order
to elute bound polypeptides, the salt concentration may be
increased.
[0235] Ion exchange chromatography may also be used as an ion
exchange separation technique. Ion exchange chromatography
separates molecules based on differences between the overall charge
of the molecules. For the purification of an antibody, the antibody
must have a charge opposite to that of the functional group
attached to the ion exchange material, e.g., resin, in order to
bind. For example, antibodies, which generally have an overall
positive charge in the buffer pH below its pI, will bind well to
cation exchange material, which contain negatively charged
functional groups.
[0236] In ion exchange chromatography, charged patches on the
surface of the solute are attracted by opposite charges attached to
a chromatography matrix, provided the ionic strength of the
surrounding buffer is low. Elution is generally achieved by
increasing the ionic strength (i.e., conductivity) of the buffer to
compete with the solute for the charged sites of the ion exchange
matrix. Changing the pH and thereby altering the charge of the
solute is another way to achieve elution of the solute. The change
in conductivity or pH may be gradual (gradient elution) or stepwise
(step elution).
[0237] Anionic or cationic substituents may be attached to matrices
in order to form anionic or cationic supports for chromatography.
Anionic exchange substituents include diethylaminoethyl(DEAE),
quaternary aminoethyl(QAE) and quaternary amine(Q) groups. Cationic
substitutents include carboxymethyl (CM), sulfoethyl(SE),
sulfopropyl(SP), phosphate(P) and sulfonate(S). Cellulose ion
exchange resins such as DE23, DE32, DE52, CM-23, CM-32 and CM-52
are available from Whatman Ltd. Maidstone, Kent, U.K.
SEPHADEX.RTM.-based and -locross-linked ion exchangers are also
known. For example, DEAE-, QAE-, CM-, and SP-SEPHADEX.RTM. and
DEAE-, Q-, CM- and S-SEPHAROSE.RTM. and SEPHAROSE.RTM. Fast Flow
are all available from Pharmacia AB. Further, both DEAE and CM
derivatized ethylene glycol-methacrylate copolymer such as
TOYOPEARL DEAE-650S or M and TOYOPEARL CM-650S or M are available
from Toso Haas Co., Philadelphia, Pa.
[0238] In one embodiment of the invention, the mixture comprising
an antibody and at least one HCP is loaded onto a cation exchange
(CEX) chromatography column The mixture is loaded onto the CEX
column in a loading buffer which may be the same as the
equilibration buffer used to equilibrate the column. As the
HCP-comprising mixture passes over the CEX column, the target
protein is adsorbed to the CEX resin and various HCPs (such as host
cell proteins, where the target protein is produced in a
recombinant host cell, or other process-derived impurities)
flowthrough or bind weakly or nonspecifically to the CEX resin. In
various embodiments, the CEX resin is a synthetic methacrylate
based polymeric resin attached to a sulfonate group (Fractogel
SO.sub.3- (Fractogel S)). In one embodiment, the equilibration
buffer comprises 20 mM Na.sub.2PO.sub.4, 25 mM NaCl, pH 6.8. Other
suitable equilibration buffers include, for example, BIS and HEPES
at physiological concentrations, for example, concentrations in the
range between about 0.5 mM and about 100 mM (e.g., 10 mM, 20 mM, 50
mM, etc.), and physiological salt concentrations (e.g., about 0.15
mM NaCl), and at pH from 5.0-9.0.
[0239] In exemplary embodiments, the CEX chromatography is a
Fractogel S column In one embodiment, about 30 gram (g) antibody
per liter (L) resin to about 40 g antibody per L resin is loaded
onto the Fractogel S column. In another embodiment, about 35 g
antibody per L resin is loaded onto the Fractogel S column at pH 7.
It has been discovered that a loading amount of about 35 g antibody
per L resin at pH 7 increases the clearance of impurities, e.g.,
HCP(s) and procathepsin L. The acceptable operating ranges of a
Fractogel S column to be used in the method of the invention are
described in Table 1 below.
TABLE-US-00001 TABLE 1 Acceptable operating ranges for Fractogel S
chromatography at pH 7 Operating parameter AOR Resin capacity
.ltoreq.35 g protein/L resin Load sample pH 6.5-7.5 Effective load
dilution 1:1-1:2 Wash 2 elution buffer 1:3-1:4 concentrate to WFI
ratio Linear velocity 75-300 cm/hr
[0240] It further has been discovered that the loading capacity of
the column can be increased by carrying out the chromatography at
pH 5. In particular, it has been discovered that a loading amount
of about 70 g antibody per L resin at pH 5 increases the clearance
of impurities, e.g., HCP(s) and procathepsin L. Accordingly, in a
pH range of about pH 5 to about pH 7, a loading amount of about 35
g to about 70 g antibody per L of resin can be used.
[0241] Following the loading of the antibody mixture onto the
column, the CEX resin is then washed with a wash buffer. In
particular, it has been discovered that a plurality of wash steps
with different wash buffers results in a further HCP-reduced
antibody preparation. Specifically, it has been discovered that
procathepsin L levels can be reduced by the use of a first wash
step and an intermediate wash step using a wash buffer and an
intermediate wash buffer, respectively. In one embodiment, the CEX
resin is first washed with a wash buffer which is the same as the
equilibration buffer. In certain embodiments, the wash buffer is 20
mM Na.sub.2PO.sub.4, 25 mM NaCl, pH 6.8. Other suitable wash
buffers include, for example, BIS and HEPES at physiological
concentrations, for example, concentration in the range between
about 0.5 mM and about 100 mM (e.g., 10 mM, 20 mM, 50 mM, etc.),
and physiological salt concentrations (e.g., about 0.15 mM NaCl),
and at pH from 5.0-9.0.
[0242] In a preferred embodiment of the invention, an intermediate
wash step is performed. It has been discovered that reduced levels
of HCP in general, and in particular, procathepsin L, can be
achieved by using an intermediate wash buffer comprising, in part,
the same buffer as the CEX elution buffer. The improved reduction
of HCP in general, and in particular procathepsin L, results in
part, from the increased conductivity in the intermediate wash
buffer. Increasing the conductivity of the intermediate wash buffer
causes charge displacement of the HCP(s), which has a lower pI
relative to the that of the antibody, thus causing the weaker
binding HCP(s) to wash through the column. Increased clearance of
the weaker binding impuities, e.g., HCP(s) including procathepsin
L, in turn provides more binding area for the target substance,
e.g., the antibody. In other embodiments, the intermediate wash
buffer contains from about 40% to about 50% elution buffer and from
about 50% to about 60% water for injection. In further embodiments,
the intermediate wash buffer contains 45% elution buffer and 55%
water for injection. In one embodiment, the wash buffer used in the
intermediate wash is 20 mM Na.sub.2PO.sub.4, 150 mM sodium
chloride, pH 7.
[0243] Following a plurality of washes, the antibody is eluted from
the first cationic exchange material such that a first eluate
having a reduced level of HCP is obtained. The first eluate also
has a reduced level of procathepsin L in view of the intermediate
wash step. In one embodiment, the first eluate obtained using the
method of the invention comprises an about 3 to an about 5 fold
decrease in HCP levels in comparison to a comparable step of
process A. In another embodiment, the first eluate obtained using
the method of the invention comprises an about 2 to an about 3 fold
decrease in cathepsin L activity in comparison to a comparable step
of process A. In one embodiment, the first eluate comprises a range
of about 90 to about 100 fold less HCP than the mixture as
determined by a HCP ELISA. In another embodiment, the first eluate
comprises cathepsin L activity ranging from between about 25 to
about 60 RFU/s/mg of antibody as measured by a cathepsin L kinetic
assay
[0244] In a preferred embodiment, the initial eluate comprising the
antibody is passed over a second IE material and a flowthrough
comprising a further reduced level of HCP is obtained. In some
embodiments, the second IE step may be batch purification as
described infra. In other embodiments, the second IE step comprises
loading the first eluate onto a second ion exchange chromatography
column, washing the column and obtaining a first flowthrough. The
second IE material may be anion exchange (AEX) resin, e.g., Q
sepharose column. In some embodiments, between about 30 g antibody
per L resin and about 40 g antibody per L or resin is loaded onto
the anion exchange column. Increasing the loading amount between
about 40 g antibody per L resin and about 50 g antibody per L resin
causes a decrease in clearance of impurities, e.g., HCP(s). As the
HCP-comprising mixture passes over the AEX column, the various
HCP(s) bind to the AEX resin, and the antibody passes through or
binds nonspecifically to the AEX resin. In certain embodiments, the
anion exchange resin is Q Sepharose.
[0245] Often, the antibody mixture to be purified will be present
in a buffer from the previous purification step. Many buffers are
available and can be selected by routine experimentation. For
example, an equilibration buffer of 25 mM trolamine, 40 mM NaCl, pH
8 may be used. In one embodiment, prior to passing the initial
eluate over the second IE material, the second IE material may be
equilibrated with equilibration buffer. This may be done, for
example, by altering the pH and conductivity of the first eluate
such that the pH and conductivity of the first eluate is
substantially similar or corresponds to the pH and conductivity of
the equilibrated second IE material, i.e., altering the pH and
conductivity of the first eluate such that it corresponds to that
of the equilibrated second ion exchange material. In some
embodiments, the pH of the AEX material (e.g. Q Sepharose) is
adjusted with equilibration buffer to range from about 7.7 to about
8.3. In further embodiments, the pH of the CEX eluate (e.g.,
initial eluate) is adjusted to range about 7.7 to about 8.3. In
certain embodiments, the pH of both the AEX material and the
initial eluate is about 8. In some embodiments, the conductivity of
the AEX material ranges from about 3.5 mS/cm to about 4.9 mS/cm. In
further embodiments, the conductivity of the initial eluate ranges
from about 3.5 mS/cm to about 4.9 mS/cm. It has been discovered
that adjustment of the load conductivity and pH to that of the
conductivity and pH of the second ion exchange material enhances
impurity clearance. In relation to process A, it has been
discovered that an overall decrease in conductivity and/or an
increase in pH of the first eluate and/or equilibrated second ion
exchange material results in increased HCP(s) clearance.
[0246] Following the loading of the antibody mixture onto the
column, the AEX resin is then washed with a wash buffer. The wash
buffer may be the same as the equilibration buffer, e.g., 25 mM
trolamine, 40 mM NaCl, pH 8. In one embodiment, the wash may be
pooled with the flowthrough such that a first flowthrough
comprising the antibody and having a reduced level of HCP is
obtained. In further embodiments, the first flowthrough has a
reduced level of procathepsin L. In one embodiment, the first
flowthrough obtained using the method of the invention comprises an
about 7 to an about 700 fold decrease in HCP levels in comparison
to a comparable step of process A. In another embodiment, the first
flowthrough obtained using the method of the invention comprises an
about 6 to an about 25 fold decrease in cathepsin L activity in
comparison to a comparable step of process A. In other embodiments,
the first flowthrough comprises a range of about 840 to about 850
fold less HCP than the first eluate as determined by a HCP ELISA.
In yet another embodiment, the first flowthrough comprises
cathepsin L activity ranging from between about 0.4 to about 4
RFU/s/mg of antibody as measured by a cathepsin L kinetic assay
[0247] Acceptable operating ranges for Q sepharose chromatography
to be used in the method of the invention are described below in
Table 2:
TABLE-US-00002 TABLE 2 Acceptable operating ranges for Q Sepharose
FF chromatography Parameter AOR Load conductivity 4.0-5.5 mS/cm
Load pH 7.8-8.2 Column loading .ltoreq.40 g/L Linear velocity
150-300 cm/hr
[0248] The use of a cationic exchange material versus an anionic
exchange material is based on the overall charge of the protein as
discussed supra. Therefore, it is within the scope of this
invention to employ an anionic exchange material prior to the use
of a cationic exchange material. Furthermore, it is within the
scope of this invention to employ only a cationic exchange material
or only an anionic exchange material.
[0249] The methods of the present invention can optionally include
further purification steps. Examples of additional purification
procedures which may be performed prior to, during, or following
the ion exchange chromatography method include fractionation on a
hydrophobic interaction chromatography (e.g. on phenyl sepharose),
ethanol precipitation, isoelectric focusing, Reverse Phase HPLC,
chromatography on silica, chromatography on heparin sepharose,
further anion exchange chromatography and/or further cation
exchange chromatography, chromatofocusing, SDS-PAGE, ammonium
sulfate precipitation, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography (e.g. using
protein A, protein G, an antibody, a specific substrate, ligand or
antigen as the capture reagent).
III.B. Hydrophobic Interaction Separation
[0250] The present invention also features methods for producing a
HCP-reduced antibody preparation from a mixture comprising an
antibody and at least one HCP further comprising a hydrophobic
interaction separation step wherein the first flowthrough is
subjected to a first hydrophobic interaction material such that a
second eluate having a reduced level of HCP is obtained.
[0251] In performing the separation, the polypeptide mixture may be
contacted with the HIC material, e.g., using a batch purification
technique or using a column. Prior to HIC purification it may be
desirable to remove any chaotropic agents or very hydrophobic
substances, e.g., by passing the mixture through a precolumn.
[0252] For example, for batch purification, HIC material is
prepared in or equilibrated to the desired equilibration buffer. A
slurry of the HIC material is obtained. The antibody solution is
contacted with the slurry to adsorb the antibody to be separated to
the HIC material. The solution comprising the HCPs that do not bind
to the HIC material is separated from the slurry, e.g., by allowing
the slurry to settle and removing the supernatant. The slurry can
be subjected to one or more washing steps. If desired, the slurry
can be contacted with a solution of lower conductivity to desorb
antibodies that have bound to the HIC material. In order to elute
bound antibodies, the salt concentration can be decreased.
[0253] In other embodiments, the hydrophobic interaction separation
step comprises loading the first flowthrough onto a column
comprising a first hydrophobic interaction material and washing the
first hydrophobic interaction material such that a second eluate is
obtained.
[0254] The hydrophobic interaction separation step may include a
hydrophobic interaction chromatography (HIC) step. Whereas ion
exchange chromatography relies on the charges of proteins, e.g.,
antibodies, to isolate them, hydrophobic interaction chromatography
uses the hydrophobic properties of some proteins, e.g., antibodies.
Hydrophobic groups on the antibody bind to hydrophillic groups on
the column. The more hydrophobic a protein is, the stronger it will
bind to the column The HIC step removes, for example, host cell
derived impurities (e.g., DNA and other high and low molecular
weight product-related species).
[0255] Hydrophobic interactions are strongest at high ionic
strength, therefore, this form of separation is conveniently
performed following salt precipitations or ion exchange procedures.
Adsorption of the antibody to a HIC column is favored by high salt
concentrations, but the actual concentrations can vary over a wide
range depending on the nature of the antibody and the particular
HIC ligand chosen. Various ions can be arranged in a so-called
soluphobic series depending on whether they promote hydrophobic
interactions (salting-out effects) or disrupt the structure of
water (chaotropic effect) and lead to the weakening of the
hydrophobic interaction. Cations are ranked in terms of increasing
salting out effect as Ba.sup.++<; Ca.sup.++<; Mg.sup.++<;
Li.sup.+<; Cs.sup.+<; Na.sup.+<; K.sup.+<;
Rb.sup.+<; NH.sub.4.sup.+, while anions may be ranked in terms
of increasing chaotropic effect as P0.sup.---<;
S0.sub.4.sup.--<; CH.sub.3COOO.sup.-<; Cl.sup.-<;
Br.sup.-<; NO.sub.3.sup.-<; ClO.sub.4.sup.-<; I.sup.-<;
SCN.sup.-
[0256] In general, Na, K or NH.sub.4 sulfates effectively promote
ligand-protein interaction in HIC. Salts may be formulated that
influence the strength of the interaction as given by the following
relationship: (NH.sub.4).sub.2SO.sub.4<; Na.sub.2SO.sub.4<;
NaCl <; NH.sub.4Cl <; NaBr <; NaSCN. In general, salt
concentrations of between about 0.75 and about 2M ammonium sulfate
or between about 1 and 4M NaCl are useful.
[0257] HIC columns normally comprise a base matrix (e.g.
cross-linked agarose or synthetic copolymer material) to which
hydrobobic ligands (e.g. alkyl or aryl groups) are coupled. The
preferred HIC column comprises an agarose resin substituted with
phenyl groups (e.g. a Phenyl Sepharose.TM. column). Many HIC
columns are available commercially. Examples include, but are not
limited to, Phenyl Sepharose.TM. 6 Fast Flow column with low or
high substitution (Pharmacia LKB Biotechnology, AB, Sweden); Phenyl
Sepharose.TM. High Performance column (Pharmacia LKB Biotechnology,
AB, Sweden); Octyl Sepharose.TM. High Performance column (Pharmacia
LKB Biotechnology, AB, Sweden); Fractogel.TM. EMD Propyl or
Fractogel.TM. EMD Phenyl columns (E. Merck, Germany);
Macro-Prep.TM. Methyl or Macro-Prep.TM. t-Butyl Supports (Bio-Rad,
California); WP HI-Propyl (C.sub.3).TM. column (J. T. Baker, New
Jersey); and Toyopearl.TM. ether, phenyl or butyl columns
(TosoHaas, PA).
[0258] Following any preliminary purification step(s), the mixture
comprising the antibody of interest and HCP(s) may be subjected to
HIC. Often, the antibody composition to be purified will be present
in a buffer from the previous purification step. However, it may be
necessary to add a buffer to the antibody composition prior to the
HIC step. Many buffers are available and can be selected by routine
experimentation. In one embodiment, the pH of the mixture
comprising the antibody to be purified and at least one HCP in a
loading buffer is adjusted to a pH of about 7 using either an acid
or base, depending on the starting pH, and a conductivity of about
136 to about 158 mS/cm. In one embodiment, the antibody mixture is
diluted with a buffer comprising 40 mM sodium phosphate, 2.2 M
(NH.sub.4).sub.2SO.sub.4, pH 7.
[0259] Prior to loading the antibody mixture, the column may be
equilibrated with equilibration buffer. In some embodiments, the
equilibration buffer is 20 mM sodium phosphate, 1.1 M
(NH.sub.4).sub.2SO.sub.4, pH 7.
[0260] In one embodiment, the mixture, e.g., first flowthrough
comprising the antibody, is loaded onto a phenyl sepharose HIC
column. In certain embodiments, the protein loading for this step
ranges between about 20 and about 40 g protein per L of resin. In
other embodiments, the protein loading for this step is about 35 g
protein per L of resin. In some embodiments, two or three
chromatography cycles may be required to process the entire
quantity of available material.
[0261] Following binding of the protein to the hydrophobic
interaction column, the column may be washed with a wash buffer
that may be the same as the equilibration buffer, e.g., 1.1 M
(NH.sub.4).sub.2SO.sub.4, pH 7.
[0262] The antibody is eluted from the column using an elution
buffer such that a second eluate is obtained. The elution buffer
can be selected using routine experimentation. The pH of the
elution buffer ranges between about 6 and about 8 and has a low
ammonium sulfate concentration (i.e., less than about 1 M
(NH.sub.4).sub.2SO.sub.4). The conductivity of the elution buffer
ranges from about 87 to about 101 mS/cm. In one embodiment, the
elution buffer contains 11 mM sodium phosphate, 0.625 M
(NH.sub.4).sub.2SO.sub.4), pH 7. It has been discovered that lower
salt concentrations result in less adalimumab binding to the resin.
The antibody is eluted from the second ion exchange material such
that a second eluate having a reduced level of HCP is obtained. The
second eluate also has a reduced level of procathepsin L. In one
embodiment, the second obtained using the method of the invention
comprises an about 10 to an about 96 fold decrease in HCP levels in
comparison to a comparable step of process A. In another
embodiment, the second eluate obtained using the method of the
invention comprises an about 5 to an about 15 fold decrease in
cathepsin L activity in comparison to a comparable step of process
A. In one embodiment, the second eluate comprises a range of about
3 to about 5 fold less HCP than the first flowthrough as determined
by a HCP ELISA. In another embodiment, the second eluate comprises
cathepsin L activity ranging from between about 0.5 to about 1.5
RFU/s/mg of antibody as measured by a cathepsin L kinetic
assay.
[0263] Acceptable operating ranges for the phenyl sepharose
chromatography column used in the methods of the invention are
shown below in Table 3.
TABLE-US-00003 TABLE 3 Acceptable operating ranges for Phenyl
Sepharose HP chromatography Operating parameter AOR Column loading
20-40 g/L Load sample dilution 0.9:1 to 1.1:1 Linear velocity
25-125 cm/hr
[0264] Further purification steps can include virus removing steps
as well as nanofiltration, ultrafiltration and/or diafiltration
steps, as described herein.
III.C. Viral Inactivation
[0265] In order to provide a margin of safety, potential undetected
viruses are inactivated during the purification process. Methods of
viral inactivation are known in the art and include heat
inactivation (pasteurization), pH inactivation, solvent/detergent
treatment, UV and gamma ray irradiation and the addition of certain
chemical inactivating agents such as .beta.-propiolactone or e.g.
copper phenanthroline as in U.S. Pat. No. 4,534,972, etc. In some
embodiments, subjecting the mixture to viral inactivation can
include pH viral inactivation. Methods of pH viral inactivation
techniques are also well known in the art. For instance, typical
methods of viral inactivation include incubating the mixture for a
period of time at low pH, subsequently neutralizing the pH and
removing particulates by filtration. The choice of pH level largely
depends on the stability profile of the antibody product and buffer
components. It is known that the quality of the target antibody
during low pH virus inactivation is affected by pH and the duration
of the low pH incubation. Virus inactivation is dependent on these
same parameters in addition to protein concentration, which may
reduce inactivation at high concentrations. Thus, the proper
parameters of protein concentration, pH and duration of
inactivation may be selected by routine experimentation.
[0266] The pH of the mixture may be lowered by any suitable acid
including, but not limited to, citric acid, acetic acid, caprylic
acid, or other suitable acids. In preferred embodiments, the pH of
the mixture is adjusted with 1 M citric acid.
[0267] In some embodiments, the mixture is incubated at pH from
about 2.9 to about 3.9 for about 15 minutes to about 180 minutes.
In further embodiments, the mixture is incubated at about pH 3.5
for about 60 minutes to about 120 minutes. In still further
embodiments, the mixture is incubated at about pH 3.5 for about 60
minutes to about 180 minutes.
[0268] In one embodiment, the mixture comprising the antibody and
HCPs is subjected to viral inactivation prior to IE separation. In
other embodiments, the initial eluate is subjected to viral
inactivation prior to IE separation. In certain embodiments, the
initial eluate is subjected to viral inactivation prior to anion
exchange chromatography.
[0269] Following viral inactivation, the mixture is adjusted as
needed for further purification steps. For example, the pH-adjusted
pool may be subjected to filtration. In one embodiment, following
low pH viral inactivation and/or filtration, the pH of the mixture
is typically adjusted to a more neutral pH, e.g., from about 6.5 to
about 8.5. For example, the mixture may be flushed with water for
injection (WFI) to obtain the desired pH.
[0270] The low pH virus inactivation parameters used in the method
of the invention are shown in Table 4 below.
TABLE-US-00004 TABLE 4 Acceptable operating parameters for low pH
virus inactivation Operating parameter AOR Incubation pH 3.0-3.7
Incubation time 60-180 min Protein concentration .ltoreq.33 g/L
[0271] The invention includes a method where the first eluate from
the ion exchange column is subjected to viral inactivation prior to
the second ion exchange chromatography step. In one embodiment,
viral inactivation is achieved through pH viral inactivation.
IV. Method for Determining Host Cell Protein (HCP) Levels
[0272] The present invention also provides methods for determining
the residual levels of Host Cell Protein (HCP) concentration in the
purified antibody composition. As described above, HCPs are
desirably excluded from the final target substance product, e.g.,
the antibody. Exemplary HCPs include proteins originating from the
source of the antibody production. Failure to identify and
sufficiently remove HCPs from the target antibody may lead to
reduced efficacy and/or adverse patient reactions.
[0273] As used herein, the term "HCP ELISA" refers to an ELISA
where the second antibody used in the assay is specific to the HCPs
produced from cells, e.g., CHO cells, used to generate the
antibody, e.g., adalimumab. The second antibody may be produced
according to conventional methods known to those of skill in the
art. For example, the second antibody may be produced using HCPs
obtained by sham production and purification runs, i.e., the same
cell line used to produce the antibody of interest is used, but the
cell line is not transfected with antibody DNA. In an exemplary
embodiment, the second antibody is produced using HPCs similar to
those expressed in the cell expression system of choice, i.e., the
cell expression system used to produce the target antibody.
[0274] Generally, HCP ELISA comprises sandwiching a liquid sample
comprising HCPs between two layers of antibodies, i.e., a first
antibody and a second antibody. The sample is incubated during
which time the HCPs in the sample are captured by the first
antibody, e.g., goat anti-CHO, affinity purified (Cygnus). A
labeled second antibody specific to the HCPs produced from the
cells used to generate the antibody, e.g., anti-CHO HCP
Biotinylated, is added, and binds to the HCPs within the sample.
The amount of HCP contained in the sample is determined using the
appropriate test based on the label of the second antibody.
[0275] HCP ELISA may be used for determining the level of HCPs in
an antibody composition, such as an eluate or flowthrough obtained
using the process described in section III above. The present
invention also provides a composition comprising an antibody,
wherein the composition has no detectable level of HCPs as
determined by an HCP Enzyme Linked Immunosorbent Assay ("ELISA").
In one embodiment, the first eluate comprises between about 12,000
to about 19,500 ng/mg of HCP. In one embodiment, the second eluate
comprises between about 1.0 and about 0.0 ng/mg of HCP.
V. Method for Determining Procathepsin L Levels
[0276] The invention provides a kinetic assay (or cathepsin L
kinetic assay) for determining the amount of procathepsin L in a
sample. Procathepsin L is a host cell protein derived from certain
expression systems and, upon activation to cathepsin L, is known to
cause fragmentation of proteins, including antibodies such as
adalimumab. Studies have demonstrated that procathepsin L is
synthesized as an inactive zymogen and later processed to the
active cathepsin L form. Activation of procathepsin L occurs by
proteolytic removal of the N-terminal pro-peptide region by either
other proteases such as cathepsin D or by autocatalytic activation
within the acidic conditions of the lysosome (Turk et al. (1999)
Eur J Biochem 259:929). Furthermore, Mason et al. (see Mason et al.
(1992) Biochem Biophysical Res Comm 189: 1659) report the
activation of cathepsin L can be achieved to a higher degree at pH
5.5 with the addition of negatively charged molecules, such as
dextran sulfate, at lower pH conditions.
[0277] Previous methods of detecting levels of procathepsin L (or
the active form cathepsin L) included analytical methods such as
weak anion exchange chromatography. Such methods are limited,
however, when testing in-process samples, i.e., samples obtained
from the process described above in section III, due to buffer
system interference and matrix effects. Thus, the invention
provides a high throughput fluorescent enzymatic method to better
monitor procathepsin L, for example, for the purpose of process
monitoring.
[0278] The kinetic assay of the invention provides a method of
determining procathepsin L at levels which cannot be readily
detected by standard end point assays. The kinetic assay also
provides a means of determining whether the level of procathepsin L
is reproducibly low. In one embodiment, samples may be obtained
from any point in the process described in Section III, in order to
confirm or determine that the level of procathepsin L is being
reduced in the overall process. Procathepsin L is activated by
removing the amino terminal from the protein. In one embodiment,
activation is achieved using a peptidase, such as, but not limited
to, cathepsin D. Once activated, cathepsin L can selectively
hydrolyze substrates. A substrate is contacted with the sample and
monitored for cathepsin L activity based on changes to the
substrate.
[0279] In a preferred embodiment, the substrate for cathepsin L
comprises a label. The label may include any agent which allows the
cathepsin activity to be determined. Examples of labeled substrates
which cathepsin L can selectively hydrolyze include synthetic
substrates such as Z-leucine-arginine-AMC(R & D Systems). The
peptide substrate may contain a fluorescent 7-amino-4-methyl
coumarin (AMC) group that is quenched by the amide bond between the
amino group of the AMC and the carboxyl group of the arginine Upon
cleavage of the amide bond by cathepsin L, the released AMC group
is fluorescent and can be measured by excitation and emission
wavelengths of 380 nm and 460 nm respectively. This excitation may
be measured and used to determine the level of cathepsin L
activity. The rate of substrate turnover is directly proportional
to the amount of cathepsin L present in the sample. This
measurement is used in combination with a reference sample having
known cathepsin L activity and known amount of cathepsin L. The
cathepsin L activity in the sample is then correlated to the amount
of antibody present in the sample. In one embodiment, the first
eluate comprises cathepsin L activity ranging from between about 25
to about 60 RFU/s/mg antibody. In another embodiment, the first
flowthrough comprises cathepsin L activity ranging from between
about 0.4 to about 4 RFU/s/mg antibody. In one embodiment, the
second eluate comprises cathepsin L activity ranging from between
about 0.5 to about 1.5 RFU/s/mg antibody.
[0280] In one embodiment, the kinetic assay comprises determining
the amount of procathepsin L in a material derived from a mammalian
cell expression system comprising by contacting the material with
an enzyme to process procathepsin L to the active cathepsin L form,
such that a cathepsin L sample is obtained. Once activated,
cathepsin L can selectively hydrolyze substrates, including
synthetic substrates such as Z-leucine-arginine-AMC. A substrate is
then added to the sample, including, for example
Z-leucine-arginine-AMC, which contains a fluorescent
7-amino-4-methyl coumarin (AMC) group that is quenched by the amide
bond between the amino group of the AMC and the carboxyl group of
the arginine. Upon cleavage of the amide bond by cathepsin L, the
released AMC group is fluorescent and can be measured by excitation
and emission wavelengths of 380 nm and 460 nm respectively. The
determined cathepsin L activity is used as an indication of the
amount of procathepsin L in the material derived from the mammalian
cell expression system, e.g., Chinese Hamster Ovary (CHO)
cells.
[0281] In one embodiment, the first eluate comprises cathepsin L
activity ranging from between about 25 to about 60 RFU/s/mg
antibody. In another embodiment, the first flowthrough comprises
cathepsin L activity ranging from between about 0.4 to about 4
RFU/s/mg antibody. In one embodiment, the second eluate comprises
cathepsin L activity ranging from between about 0.5 to about 1.5
RFU/s/mg antibody.
[0282] The invention also encompasses ranges intermediate to the
above recited amounts are also intended to be part of this
invention. For example, ranges of values using a combination of any
of the above recited values as upper and/or lower limits are
intended to be included, as well as any number between the
described range.
[0283] The invention includes any of the above-mentioned
modifications, alone or in combination with one another.
VI. Pharmaceutical Compositions
[0284] Antibodies obtained using the process of the invention may
be incorporated into pharmaceutical compositions suitable for
administration to a subject. Typically, the pharmaceutical
composition comprises an antibody, or antigen-binding portion
thereof, and a pharmaceutically acceptable carrier. As used herein,
"pharmaceutically acceptable carrier" includes any and all
solvents, dispersion media, coatings, antibacterial and antifungal
agents, isotonic and absorption delaying agents, and the like that
are physiologically compatible. Examples of pharmaceutically
acceptable carriers include one or more of water, saline, phosphate
buffered saline, dextrose, glycerol, ethanol and the like, as well
as combinations thereof. In many cases, it is preferable to include
isotonic agents, for example, sugars, polyalcohols such as
mannitol, sorbitol, or sodium chloride in the composition.
Pharmaceutically acceptable carriers may further comprise minor
amounts of auxiliary substances such as wetting or emulsifying
agents, preservatives or buffers, which enhance the shelf life or
effectiveness of antibody, or antigen-binding portion thereof.
[0285] Pharmaceutical compositions comprising antibodies, or
antigen-binding portions thereof, purified using the methods of the
invention may be found in a variety of forms. These include, for
example, liquid, semi-solid and solid dosage forms, such as liquid
solutions (e.g., injectable and infusible solutions), dispersions
or suspensions, tablets, pills, powders, liposomes and
suppositories. The preferred form depends on the intended mode of
administration and therapeutic application. Typical preferred
compositions are in the form of injectable or infusible solutions,
such as compositions similar to those used for passive immunization
of humans with other antibodies or other TNF.alpha. inhibitors. The
preferred mode of administration is parenteral (e.g., intravenous,
subcutaneous, intraperitoneal, intramuscular). In a preferred
embodiment, the antibody is administered by intravenous infusion or
injection. In another preferred embodiment, the antibody is
administered by intramuscular or subcutaneous injection.
[0286] Therapeutic compositions typically must be sterile and
stable under the conditions of manufacture and storage. The
composition can be formulated as a solution, microemulsion,
dispersion, liposome, or other ordered structure suitable to high
drug concentration. Sterile injectable solutions can be prepared by
incorporating the active compound (i.e., antibody, or
antigen-binding portion thereof) in the required amount in an
appropriate solvent with one or a combination of ingredients
enumerated above, as required, followed by filtered sterilization.
Generally, dispersions are prepared by incorporating the active
compound into a sterile vehicle that contains a basic dispersion
medium and the required other ingredients from those enumerated
above. In the case of sterile powders for the preparation of
sterile injectable solutions, the preferred methods of preparation
are vacuum drying and freeze-drying that yields a powder of the
active ingredient plus any additional desired ingredient from a
previously sterile-filtered solution thereof. The proper fluidity
of a solution can be maintained, for example, by the use of a
coating such as lecithin, by the maintenance of the required
particle size in the case of dispersion and by the use of
surfactants. Prolonged absorption of injectable compositions can be
brought about by including in the composition an agent that delays
absorption, for example, monostearate salts and gelatin.
[0287] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, an antibody, or
antigen-binding portion thereof, for use in the methods of the
invention is coformulated with and/or coadministered with one or
more additional therapeutic agents. For example, an
anti-hTNF.alpha. antibody or antibody portion of the invention may
be coformulated and/or coadministered with one or more DMARD or one
or more NSAID or one or more additional antibodies that bind other
targets (e.g., antibodies that bind other cytokines or that bind
cell surface molecules), one or more cytokines, soluble TNF.alpha.
receptor (see e.g., PCT Publication No. WO 94/06476) and/or one or
more chemical agents that inhibit hTNF.alpha. production or
activity (such as cyclohexane-ylidene derivatives as described in
PCT Publication No. WO 93/19751) or any combination thereof.
Furthermore, one or more antibodies of the invention may be used in
combination with two or more of the foregoing therapeutic agents.
Such combination therapies may advantageously utilize lower dosages
of the administered therapeutic agents, thus avoiding possible side
effects, complications or low level of response by the patient
associated with the various monotherapies.
[0288] In one embodiment, the invention includes pharmaceutical
compositions comprising an effective amount of a TNF.alpha.
antibody, or antigen-binding portion thereof, and a
pharmaceutically acceptable carrier, wherein the effective amount
of the TNF.alpha. antibody may be effective to treat a
TNF.alpha.-related disorder, including, for example, Crohn's
disease. In one embodiment, the antibody or antibody portion is
incorporated into a pharmaceutical formulation as described in
PCT/IB03/04502 and U.S. application Ser. No. 10/222,140,
incorporated by reference herein. This formulation includes a
concentration 50 mg/ml of the antibody adalimumab, wherein one
pre-filled syringe contains 40 mg of antibody for subcutaneous
injection.
[0289] The antibodies, or antibody-portions, obtained using the
methods of the present invention can be administered by a variety
of methods known in the art, although for many therapeutic
applications, the preferred route/mode of administration is
subcutaneous injection. In another embodiment, administration is
via intravenous injection or infusion. As will be appreciated by
the skilled artisan, the route and/or mode of administration will
vary depending upon the desired results. In certain embodiments,
the active compound may be prepared with a carrier that will
protect the compound against rapid release, such as a controlled
release formulation, including implants, transdermal patches, and
microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate,
polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and
polylactic acid. Many methods for the preparation of such
formulations are patented or generally known to those skilled in
the art. See, e.g., Sustained and Controlled Release Drug Delivery
Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York,
1978.
[0290] The antibodies, or antigen-binding portion thereof, obtained
using the methods of the invention can also be administered in the
form of protein crystal formulations which include a combination of
protein crystals encapsulated within a polymeric carrier to form
coated particles. The coated particles of the protein crystal
formulation may have a spherical morphology and be microspheres of
up to 500 micro meters in diameter or they may have some other
morphology and be microparticulates. The enhanced concentration of
protein crystals allows the antibody of the invention to be
delivered subcutaneously. In one embodiment, the antibodies of the
invention are delivered via a protein delivery system, wherein one
or more of a protein crystal formulation or composition, is
administered to a subject with a TNF.alpha.-related disorder.
Compositions and methods of preparing stabilized formulations of
whole antibody crystals or antibody fragment crystals are also
described in WO 02/072636, which is incorporated by reference
herein. In one embodiment, a formulation comprising the
crystallized antibody fragments described in PCT/IB03/04502 and
U.S. application Ser. No. 10/222,140, incorporated by reference
herein, are used to treat a TNF.alpha.-related disorder using the
multiple-variable dose methods of the invention.
[0291] In certain embodiments, an antibodies, or antigen-binding
portion thereof, obtained using the methods of the invention may be
orally administered, for example, with an inert diluent or an
assimilable edible carrier. The compound (and other ingredients, if
desired) may also be enclosed in a hard or soft shell gelatin
capsule, compressed into tablets, or incorporated directly into the
subject's diet. For oral therapeutic administration, the compounds
may be incorporated with excipients and used in the form of
ingestible tablets, buccal tablets, troches, capsules, elixirs,
suspensions, syrups, wafers, and the like. To administer a compound
of the invention by other than parenteral administration, it may be
necessary to coat the compound with, or co-administer the compound
with, a material to prevent its inactivation.
[0292] The pharmaceutical compositions of the invention may include
a "therapeutically effective amount" or a "prophylactically
effective amount" of an antibody or or antigen-binding portion
thereof of the invention. A "therapeutically effective amount"
refers to an amount effective, at dosages and for periods of time
necessary, to achieve the desired therapeutic result. A
therapeutically effective amount of the antibody, or
antigen-binding portion thereof, may vary according to factors such
as the disease state, age, sex, and weight of the individual, and
the ability of the antibody, antibody portion, other TNF.alpha.
inhibitor to elicit a desired response in the individual. A
therapeutically effective amount is also one in which any toxic or
detrimental effects of the antibody, or antigen-binding portion
thereof, are outweighed by the therapeutically beneficial effects.
A "prophylactically effective amount" refers to an amount
effective, at dosages and for periods of time necessary, to achieve
the desired prophylactic result. Typically, since a prophylactic
dose is used in subjects prior to or at an earlier stage of
disease, the prophylactically effective amount will be less than
the therapeutically effective amount.
[0293] Dosage regimens may be adjusted to provide the optimum
desired response (e.g., a therapeutic or prophylactic response).
For example, a single bolus may be administered, several divided
doses may be administered over time or the dose may be
proportionally reduced or increased as indicated by the exigencies
of the therapeutic situation. It is especially advantageous to
formulate parenteral compositions in dosage unit form for ease of
administration and uniformity of dosage. Dosage unit form as used
herein refers to physically discrete units suited as unitary
dosages for the mammalian subjects to be treated; each unit
comprising a predetermined quantity of active compound calculated
to produce the desired therapeutic effect in association with the
required pharmaceutical carrier. The specification for the dosage
unit forms of the invention are dictated by and directly dependent
on (a) the unique characteristics of the active compound and the
particular therapeutic or prophylactic effect to be achieved, and
(b) the limitations inherent in the art of compounding such an
active compound for the treatment of sensitivity in
individuals.
[0294] An exemplary, non-limiting range for a therapeutically or
prophylactically effective amount of an antibody, or
antigen-binding portion thereof, is 10 to 200 mg, more preferably
20 to 160 mg, more preferably 40 to 80 mg, and most preferably 80
mg. In one embodiment, the therapeutically effective amount of an
antibody or, antigen-binding portion thereof, is about 20 mg. In
another embodiment, the therapeutically effective amount of an
antibody or portion thereof is about 40 mg. In still another
embodiment, the therapeutically effective amount of an antibody or,
antigen-binding portion thereof, is about 80 mg. In one embodiment,
the therapeutically effective amount of an antibody or portion
thereof for use in the methods of the invention is about 120 mg. In
yet another embodiment, the therapeutically effective amount of an
antibody, or antigen-binding portion thereof, is about 160 mg.
Ranges intermediate to the above recited dosages, e.g. about 78.5
to about 81.5; about 15 to about 25; about 30 to about 50; about 60
to about 100; about 90 to about 150; about 120 to about 200, are
also intended to be part of this invention. For example, ranges of
values using a combination of any of the above recited values as
upper and/or lower limits are intended to be included.
[0295] It is to be noted that dosage values may vary with the type
and severity of the condition to be alleviated. It is to be further
understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that dosage
ranges set forth herein are exemplary only and are not intended to
limit the scope or practice of the claimed composition.
[0296] Antibodies, or antibody-portions thereof, obtained using the
methods of the invention may be administered on a biweekly dosing
regimen as described in WO 02/100330, a low dose regimen as
described in WO 04/037205, and a multiple variable dosing regimen
as described in WO 05/110452, each of which is incorporated by
reference herein.
[0297] The invention also pertains to packaged pharmaceutical
compositions, articles of manufacture, or kits comprising the
antibody, or antigen-binding portion thereof, obtained using the
process of the invention. The article of manufacture may comprise
an antibody, or antigen-binging portion thereof, obtained using the
method of the invention and packaging material. The article of
manufacture may also comprise label or package insert indicating
the formulation or composition comprising the antibody, or
antigen-binding portion thereof, has a reduced level of HCP and/or
procathepsin L. The article of manufacture may comprise a label or
package insert contained within the packaging material indicating
that the adalimumab formulation comprises no greater than about 70
ng/mg of HCP or a label or package insert contained within the
packaging material indicating that the adalimumab formulation
comprises no greater than about 13 ng/mg. The article of
manufacture may comprise a label or package insert contained within
the packaging material indicating that the adalimumab formulation
comprises no greater than about 5 ng HCP/mg adalimumab. The article
of manufacture may also comprise packaging material indicating that
the adalimumab formulation comprises no greater a level of
procathepsin L than that indicated by a cathepsin L activity of
about 3.0 RFU/s/mg adalimumab.
VII. Methods of Treatment
[0298] The invention a method of producing an HCP- or procathepsin
L-reduced antibody preparation which can be used for inhibiting
TNF.alpha. activity in a subject suffering from a disorder in which
TNF.alpha. activity is detrimental. TNF.alpha. has been implicated
in the pathophysiology of a wide variety of disorders (see e.g.,
Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Pat. No.
5,231,024 to Moeller et al.; European Patent Publication No. 260
610 B1 by Moeller, A.). TNF.alpha. has been implicated in the
pathophysiology of a wide variety of a TNF.alpha.-related disorders
including sepsis, infections, autoimmune diseases, transplant
rejection and graft-versus-host disease (see e.g., Moeller, A., et
al. (1990) Cytokine 2:162-169; U.S. Pat. No. 5,231,024 to Moeller
et al.; European Patent Publication No. 260 610 B1 by Moeller, A.,
et al. Vasilli, P. (1992) Annu. Rev. Immunol. 10:411-452; Tracey,
K. J. and Cerami, A. (1994) Annu. Rev. Med. 45:491-503). The
invention a method of producing an HCP- or procathepsin L-reduced
antibody preparation methods which are beneficial for inhibiting
TNF.alpha. activity in a subject suffering from a
TNF.alpha.-related disorder, which method comprises administering
to a subject an initial induction dose and subsequently
administering a treatment dose of an antibody, or antigen-binding
fragment thereof, such that TNF.alpha. activity is inhibited.
Preferably, the TNF.alpha. is human TNF.alpha. and the subject is a
human subject. In one embodiment, the TNF.alpha. inhibitor is
adalimumab, also referred to as HUMIRA.RTM. (D2E7).
[0299] As used herein, the term "a disorder in which TNF.alpha.
activity is detrimental" is intended to include diseases and other
disorders in which the presence of TNF.alpha. in a subject
suffering from the disorder has been shown to be or is suspected of
being either responsible for the pathophysiology of the disorder or
a factor that contributes to a worsening of the disorder.
Accordingly, a disorder in which TNF.alpha. activity is detrimental
is a disorder in which inhibition of TNF.alpha. activity is
expected to alleviate the symptoms and/or progression of the
disorder. Such disorders may be evidenced, for example, by an
increase in the concentration of TNF.alpha. in a biological fluid
of a subject suffering from the disorder (e.g., an increase in the
concentration of TNF.alpha. in serum, plasma, synovial fluid, etc.
of the subject), which can be detected, for example, using an
anti-TNF.alpha. antibody as described above. There are numerous
examples of disorders in which TNF.alpha. activity is detrimental.
The use of TNF.alpha. antibodies and antibody portions obtained
using methods of the invention for the treatment of specific
disorders is discussed further below:
A. Sepsis
[0300] Tumor necrosis factor has an established role in the
pathophysiology of sepsis, with biological effects that include
hypotension, myocardial suppression, vascular leakage syndrome,
organ necrosis, stimulation of the release of toxic secondary
mediators and activation of the clotting cascade (see e.g.,
Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Pat. No.
5,231,024 to Moeller et al.; European Patent Publication No. 260
610 B1 by Moeller, A.; Tracey, K. J. and Cerami, A. (1994) Annu.
Rev. Med. 45:491-503; Russell, D. and Thompson, R. C. (1993) Curr.
Opin. Biotech. 4:714-721). The multiple-variable dose methods of
the invention can be used to treat sepsis in any of its clinical
settings, including septic shock, endotoxic shock, gram negative
sepsis and toxic shock syndrome.
[0301] Furthermore, to treat sepsis, an anti-hTNF.alpha. antibody,
or antibody portion, obtained using the process of the invention
can be coadministered with one or more additional therapeutic
agents that may further alleviate sepsis, such as an interleukin-1
inhibitor (such as those described in PCT Publication Nos. WO
92/16221 and WO 92/17583), the cytokine interleukin-6 (see e.g.,
PCT Publication No. WO 93/11793) or an antagonist of platelet
activating factor (see e.g., European Patent Application
Publication No. EP 374 510). In a preferred embodiment, an
anti-TNF.alpha. antibody or antibody portion is administered to a
human subject within a subgroup of sepsis patients having a serum
or plasma concentration of IL-6 above 500 pg/ml, and more
preferably 1000 pg/ml, at the time of treatment (see PCT
Publication No. WO 95/20978 by Daum, L., et al.).
B. Autoimmune Diseases
[0302] Tumor necrosis factor has been implicated in playing a role
in the pathophysiology of a variety of autoimmune diseases. For
example, TNF.alpha. has been implicated in activating tissue
inflammation and causing joint destruction in rheumatoid arthritis
(see e.g., Moeller, A., et al. (1990) Cytokine 2:162-169; U.S. Pat.
No. 5,231,024 to Moeller et al.; European Patent Publication No.
260 610 B1 by Moeller, A.; Tracey and Cerami, supra; Arend, W. P.
and Dayer, J-M. (1995) Arth. Rheum. 38:151-160; Fava, R. A., et al.
(1993) Clin. Exp. Immunol. 94:261-266). TNF.alpha. also has been
implicated in promoting the death of islet cells and in mediating
insulin resistance in diabetes (see e.g., Tracey and Cerami, supra;
PCT Publication No. WO 94/08609). TNF.alpha. also has been
implicated in mediating cytotoxicity to oligodendrocytes and
induction of inflammatory plaques in multiple sclerosis (see e.g.,
Tracey and Cerami, supra). TNF.alpha. also has been implicated in
mediating cytotoxicity to oligodendrocytes and induction of
inflammatory plaques in multiple sclerosis (see e.g., Tracey and
Cerami, supra). Chimeric and humanized murine anti-hTNF.alpha.
antibodies have undergone clinical testing for treatment of
rheumatoid arthritis (see e.g., Elliott, M. J., et al. (1994)
Lancet 344:1125-1127; Elliot, M. J., et al. (1994) Lancet
344:1105-1110; Rankin, E. C., et al. (1995) Br. J. Rheumatol.
34:334-342).
[0303] TNF.alpha. antibodies, such as adalimumab, may be used to
treat autoimmune diseases, in particular those associated with
inflammation. Examples of such autoimmune conditions include
rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis and
gouty arthritis, allergy, multiple sclerosis, autoimmune diabetes,
autoimmune uveitis and nephrotic syndrome. Other examples of
autoimmune conditions include multisystem autoimmune diseases and
autoimmune hearing loss.
[0304] Typically, the antibody, or antibody portion, is
administered systemically, although for certain disorders, local
administration of the antibody or antibody portion at a site of
inflammation may be beneficial (e.g., local administration in the
joints in rheumatoid arthritis or topical application to diabetic
ulcers, alone or in combination with a cyclohexane-ylidene
derivative as described in PCT Publication No. WO 93/19751).
TNF.alpha. inhibitors, including human antibodies, and antibody
portions such as D2E7, also can be administered with one or more
additional therapeutic agents useful in the multiple-variable dose
treatment of autoimmune diseases, as discussed further below.
[0305] In one embodiment of the invention, a TNF.alpha. antibody
obtained using the methods of the invention is used to treat
autoimmune disorders such as lupus. Lupus is has been shown to be
associated with TNF activity (Shvidel et al. (2002) Hematol J.
3:32; Studnicka-Benke et al. (1996) Br J. Rheumatol. 35:1067). The
term "lupus" as used herein refers to a chronic, inflammatory
autoimmune disorder called lupus erythematosus that may affect many
organ systems including the skin, joints and internal organs. Lupus
is a general term which includes a number of specific types of
lupus, including systemic lupus, lupus nephritis, and lupus
cerebritis. In systemic lupus (SLE), the body's natural defenses
are turned against the body and rogue immune cells attack the
body's tissues. Antibodies may be produced that can react against
the body's blood cells, organs, and tissues. This reaction leads to
immune cells attacking the affected systems, producing a chronic
disease. Lupus nephritis, also referred to as lupus glomerular
disease, is kidney disorder that is usually a complication of SLE,
and is characterized by damage to the glomerulus and progressive
loss of kidney function. Lupus cerebritis refers to another
complication of SLE, which is inflammation of the brain and/or
central nervous system.
[0306] Another autoimmune disease which can be treated using a
TNF.alpha. antibody is Crohn's disease, which is described in more
detail below in the Intestinal Disorders Section.
C. Infectious Diseases
[0307] Tumor necrosis factor has been implicated in mediating
biological effects observed in a variety of infectious diseases.
For example, TNF.alpha. has been implicated in mediating brain
inflammation and capillary thrombosis and infarction in malaria.
TNF.alpha. also has been implicated in mediating brain
inflammation, inducing breakdown of the blood-brain barrier,
triggering septic shock syndrome and activating venous infarction
in meningitis. TNF.alpha. also has been implicated in inducing
cachexia, stimulating viral proliferation and mediating central
nervous system injury in acquired immune deficiency syndrome
(AIDS). Accordingly, antibodies, and antibody portions, directed
against TNF, can be used for treatment of infectious diseases,
including bacterial meningitis (see e.g., European Patent
Application Publication No. EP 585 705), cerebral malaria, AIDS and
AIDS-related complex (ARC) (see e.g., European Patent Application
Publication No. EP 230 574), as well as cytomegalovirus infection
secondary to transplantation (see e.g., Fietze et al. (1994)
Transplantation 58:675). The antibodies, and antibody portions, of
the invention, also can be used to alleviate symptoms associated
with infectious diseases, including fever and myalgias due to
infection (such as influenza) and cachexia secondary to infection
(e.g., secondary to AIDS or ARC).
D. Transplantation
[0308] Tumor necrosis factor has been implicated as a key mediator
of allograft rejection and graft versus host disease (GVHD) and in
mediating an adverse reaction that has been observed when the rat
antibody OKT3, directed against the T cell receptor CD3 complex, is
used to inhibit rejection of renal transplants (see e.g., Eason et
al. (1995) Transplantation 59:300; Suthanthiran and Strom (1994)
New Engl. J. Med. 331:365). Accordingly, the antibodies, and
antibody portions, of the invention, can be used to inhibit
transplant rejection using multiple-variable dose treatment,
including rejections of allografts and xenografts and to inhibit
GVHD. Although the antibody or antibody portion may be used alone,
more preferably it is used in combination with one or more other
agents that inhibit the immune response against the allograft or
inhibit GVHD. For example, in one embodiment, an antibody or
antibody portion of the invention is used in combination with OKT3
to inhibit OKT3-induced reactions. In another embodiment, an
antibody or antibody portion of the invention is used in
combination with one or more antibodies directed at other targets
involved in regulating immune responses, such as the cell surface
molecules CD25 (interleukin-2 receptor-.alpha.), CD11a (LFA-1),
CD54 (ICAM-1), CD4, CD45, CD28/CTLA4, CD80 (B7-1) and/or CD86
(B7-2). In yet another embodiment, an antibody or antibody portion
of the invention is used in combination with one or more general
immunosuppressive agents, such as cyclosporin A or FK506.
E. Malignancy
[0309] Tumor necrosis factor has been implicated in inducing
cachexia, stimulating tumor growth, enhancing metastatic potential
and mediating cytotoxicity in malignancies. Accordingly,
antibodies, and antibody portions, which directed against TNF, can
be used in the treatment of malignancies, wherein treatment
inhibits tumor growth or metastasis and/or alleviates cachexia
secondary to malignancy. The antibody, or antibody portion, may be
administered systemically or locally to the tumor site.
F. Pulmonary Disorders
[0310] Tumor necrosis factor has been implicated in the
pathophysiology of adult respiratory distress syndrome (ARDS),
including stimulating leukocyte-endothelial activation, directing
cytotoxicity to pneumocytes and inducing vascular leakage syndrome.
The antibody obtained using the methods of the invention may be
used to treat various pulmonary disorders, including adult
respiratory distress syndrome (see e.g., PCT Publication No. WO
91/04054), shock lung, chronic pulmonary inflammatory disease,
pulmonary sarcoidosis, pulmonary fibrosis and silicosis. The
antibody, or antibody portion, may be administered systemically or
locally to the lung surface, for example as an aerosol. An
antibody, or antibody portion, also can be administered with one or
more additional therapeutic agents useful in the treatment of
pulmonary disorders, as discussed further below.
[0311] Other examples of pulmonary disorders in which TNF.alpha.
has been implicated in the pathophysiology include idiopathic
interstitial lung disease and chronic obstructive airway disorders
(see e.g., Piquet et al. (1989) J Exp Med. 170:655; Whyte et al.
(2000) Am J Respir Crit Care Med. 162:755; Anticevich et al. (1995)
Eur J. Pharmacol. 284: 221). The invention further provides methods
for treating TNF.alpha. activity in a subject suffering from such a
pulmonary disorder, which method comprises administering to the
subject an antibody, or antibody portion, such that TNF.alpha.
activity in the subject suffering from idiopathic interstitial lung
disease or a chronic obstructive airway disorder is inhibited.
Examples of idiopathic interstitial lung diseases and chronic
obstructive airway disorders in which TNF.alpha. activity is
detrimental are discussed further below.
[0312] 1. Idiopathic Interstitial Lung Disease
[0313] In one embodiment, the TNF.alpha. antibody obtained using
the method of the invention is used to treat subjects who have an
idiopathic interstitial lung disease. The term "idiopathic
pulmonary fibrosis" or "IPF" refers to a group of disorders
characterized by inflammation and eventually scarring of the deep
lung tissues, leading to shortness of breath. The scarring of the
alveoli (air sacs) and their supporting structures (the
interstitium) in IPF eventually leads to a loss of the functional
alveolar units and a reduction of the transfer of oxygen from air
to blood. IPF is also referred to as diffuse parenchymal lung
disease; alveolitis; cryptogenic fibrosing alveolitis (CFA);
idiopathic pulmonary pneumonitis (IPP); and usual interstitial
pneumonitis (UIP). IPF is often used synonymously with UIP
("IPF/UIP") because UIP is the most common cellular pattern seen in
the pathologic diagnosis of IPF.
[0314] Idiopathic interstitial lung diseases affect the lungs in
three ways: first, the lung tissue is damaged in some known or
unknown way; second, the walls of the air sacs in the lung become
inflamed; and finally, scarring (or fibrosis) begins in the
interstitium (or tissue between the air sacs), and the lung becomes
stiff. Examples of idiopathic interstitial lung diseases include
idiopathic pulmonary fibrosis (IPF). Tumor necrosis factor has been
implicated in the pathophysiology of idiopathic pulmonary fibrosis
(IPF) (see Piquet et al. (1989) J Exp Med. 170:655; Whyte et al.
(2000) Am J Respir Crit Care Med 162:755 Corbett et al. (2002) Am J
Respir Crit Care Med. 165:690). For example, it has been found that
IPF patients have increased levels of TNF expression in macrophages
and in type II epithelial cells (Piquet et al. (1993) Am J Pathol
143:651; Nash et al. (1993) Histopathology 22:343; Zhang et al.
(1993) J Immunol 150:4188). Certain genetic polymorphisms are also
associated with increased TNF expression, and are implicated in
playing a role in IPF and silicosis (Whyte et al., supra; Corbett
et al., supra).
[0315] Patients with IPF often exhibit certain symptoms, including
a dry cough, chest pain, and/or shortness of breath. Commonly used
drugs for the treatment of IPF are prednisone and cytoxan, although
only a fraction of patients improve with continued use of these
drugs (American Thoracic Society (2000) Am. J. Respir. Crit. Care
Med. 161:646). Oxygen administration and transplantation of the
lung are other choices for treatment. In one embodiment, antibodies
obtained through the methods of the invention may be used in
combination with another therapeutic agent, for example oxygen, for
the treatment of idiopathic pulmonary fibrosis.
[0316] Examples of animal models used to study idiopathic
interstitial lung disease and chronic obstructive airway disorders
include ovalbumin (OVA) induced allergic asthma mice and cigarette
smoke induced chronic obstructive pulmonary disease mice (see
Hessel et al. (1995) Eur J Pharmacol. 293:401; Keast et al. (1981)
J. Pathol. 135:249).
[0317] 2. Chronic Obstructive Airway Disorder
[0318] In one embodiment, a TNF.alpha. antibody is used to treat a
subject who has a chronic obstructive airflow disorder. In these
diseases, airflow obstruction may be chronic and persistent or
episodic and recurrent. Airflow obstruction is usually determined
by forced expiratory spirometry, which is the recording of exhaled
volume against time during a maximal expiration. In a subject who
does not have an obstructed airflow, a full forced expiration
usually takes between 3 and 4 seconds. In a patient with chronic
obstructive airflow disorder, wherein airflow is obstructed, it
usually takes up to 15 to 20 seconds and may be limited by
breath-holding time. The normal forced expiratory volume in the
first second of expiration (FEV.sub.1) is easily measured and
accurately predicted on the basis of age, sex, and height. The
ratio of FEV.sub.1 to forced vital capacity (FEV.sub.1/FVC)
normally exceeds 0.75. Recording airflow against volume during
forced expiration and a subsequent forced inspiration--the
flow-volume loop--is also useful, mainly for distinguishing upper
from lower airway narrowing. Examples of chronic obstructive airway
disorders are described below.
[0319] a. Asthma
[0320] Tumor necrosis factor has been implicated in the
pathophysiology of asthma, (Anticevich et al. (1995) Eur J.
Pharmacol. 284:221; Thomas et al. 1995. Am J Respir Crit Care Med.
152:76; Thomas and Heywood (2002) Thorax. 57:774). For example,
acute asthma attacks have been found to be associated with
pulmonary neutrophilia and elevated BAL TNF levels (Ordonez et al.
(2000) Am J Respir Crit Care Med 161:1185). It has been found that
the severity of asthma symptoms correlates with endotoxin levels in
house dust. In rats, anti-TNF antibodies reduced endotoxin-induced
airway changes (Kips et al. (1992) Am Rev Respir Dis 145:332).
[0321] The term "asthma" as used herein, refers to a disorder in
which inflammation of the airways causes airflow into and out of
the lungs to be restricted. Asthma is also referred to as bronchial
asthma, exercise induced asthma--bronchial, and reactive airways
disease (RAD). In some instances, asthma is associated with
allergies and/or is familial. Asthma includes a condition which is
characterized by widespread fluctuations in the diameter or caliber
of bronchial airways over short periods of time, resulting in
changes in lung function. The resulting increased resistance to air
flow produces symptoms in the affected subject, including
breathlessness (dyspnea), chest constriction or "tightness," and
wheezing.
[0322] Patients with asthma are characterized according to NIH
guidelines, are described as mild intermittent, mild persistent,
moderate persistent, and severe persistent (see NAEPP Expert Panel
Report Guidelines for the Diagnosis and Management of Asthma-Update
on Selected Topics 2002. JACI-2002; 110: S141-S209; Guidelines for
the Diagnosis and Management of Asthma. NIH Publication 97-4051,
July 1997). Patients diagnosed with moderate persistent asthma are
often treated with inhaled corticosteroids. Patients diagnosed with
severe persistent asthma are often treated with high dose inhaled
corticosteroids and p.o. corticosteroids.
[0323] b. Chronic Obstructive Pulmonary Disease (COPD)
[0324] Tumor necrosis factor has been implicated in the
pathophysiology of chronic obstructive pulmonary disease, (Keatings
(2000) Chest. 118:971; Sakao et al. (2001) Am Respir Crit Care Med.
163:420; Sakao et al. (2002) Chest. 122:416). The term "chronic
obstructive pulmonary disease" or "COPD" as used interchangeably
herein, refers to a group of lung diseases characterized by limited
airflow with variable degrees of air sack enlargement and lung
tissue destruction. The term COPD includes chronic bronchitis
(mucous hypersecretion with goblet cell submucosal gland
hyperplasia), chronic obstructive bronchitis, or emphysema
(destruction of airway parenchyma), or combinations of these
conditions. Emphysema and chronic bronchitis are the most common
forms of chronic obstructive pulmonary disease. COPD is defined by
irreversible airflow obstruction.
[0325] In COPD, chronic inflammation leads to fixed narrowing of
small airways and lung parenchyma and alveolar wall destruction
(emphysema). This is characterized by increased numbers of alveolar
macrophages, neutrophils, and cytotoxic T lymphocytes, and the
release of multiple inflammatory mediators (lipids, chemokines,
cytokines, growth factors). This inflammation leads to fibrosis
with a narrowing of the small airways and lung parenchymal
destruction. There is also a high level of oxidative stress, which
may amplify this inflammation.
G. Intestinal Disorders
[0326] Tumor necrosis factor has been implicated in the
pathophysiology of inflammatory bowel disorders including Crohn's
disease (see e.g., Tracy et al. (1986) Science 234:470; Sun et al.
(1988) J. Clin. Invest. 81:1328; MacDonald et al. (1990) Clin. Exp.
Immunol. 81:301). Chimeric murine anti-hTNF.alpha. antibodies have
undergone clinical testing for treatment of Crohn's disease (van
Dullemen et al. (1995) Gastroenterology 109:129). The invention
includes treatment comprising administering a TNF.alpha. antibody
obtained using the method of the invention to treat intestinal
disorders, such as idiopathic inflammatory bowel disease, using
human antibodies, or antigen-binding fragments thereof. Idiopathic
inflammatory bowel disease includes two syndromes, Crohn's disease
and ulcerative colitis. In one embodiment, an antibody obtained
using the method of the invention is also used to treat disorders
often associated with IBD and Crohn's disease. The term
"inflammatory bowel disorder (IBD)-related disorder" or "Crohn's
disease-related disorder," as used interchangeably herein, is used
to describe conditions and complications commonly associated with
IBD and Crohn's disease.
[0327] The invention includes a multiple-variable dose regimen
comprising administering a TNF.alpha. antibody to treat Crohn's
disease. The treatment of Crohn's disease is based on location,
extent, and severity of disease. Pharmacologic interventions
include anti-inflammatory agents (aminosalicylates and
corticosteroids) and immunomodulatory agents (azathioprine and
6-mercaptopurine [6-MP], cyclosporine, methotrexate [MTX],
antibiotic agents, and biologic agents).C-reactive protein (CRP)
and erythrocyte sedimentation rate (ESR) levels reflect
non-specific acute phase reactions. Endoscopy is a primary means of
diagnosing Crohn's disease. Radiologic features of Crohn's disease
are shown by barium examination includes mucosal edema, aphthous
and linear ulcerations, asymmetrical narrowing and strictures, and
separation of adjacent loops of bowel caused by mesenteric
thickening. Abnormalities are focal and asymmetric. The primary
histologic lesion is anaphthous ulcer. Subjects with Crohn's
disease can be evaluated using the Crohn's Disease Activity Index
(CDAI), which is a standard measure of the severity of the disease
with higher scores indicating more severe disease activity.
[0328] Examples of Crohn's disease-related disorders which can be
treated using the methods of the invention include fistulas in the
bladder, vagina, and skin; bowel obstructions; abscesses;
nutritional deficiencies; complications from corticosteroid use;
inflammation of the joints; erythem nodosum; pyoderma gangrenosum;
and lesions of the eye. Other disorders commonly associated with
Crohn's disease include Crohn's-related arthralgias, fistulizing
Crohn's, indeterminant colitis, and pouchitis.
H. Cardiac Disorders
[0329] An antibody, or antigen-binding fragment thereof, obtained
using the method of the invention also can be used to treat in of
various cardiac or coronary disorders, including ischemia of the
heart (see e.g., European Patent Application Publication No. EP 453
898) and heart insufficiency (weakness of the heart muscle)(see
e.g., PCT Publication No. WO 94/20139). TNF.alpha. has also been
implicated in the pathophysiology of restenosis (see e.g., Clausell
et al. (1994), supra; Medall et al. (1997) Heart 78:273).
[0330] As used herein, the term "a cardiac disorder in which
TNF.alpha. activity is detrimental" is intended to include coronary
and cardiovascular diseases in which the presence of TNF.alpha. in
a subject suffering from the disorder has been shown to be or is
suspected of being either responsible for the pathophysiology of
the disorder or a factor that contributes to a worsening of the
disorder, including cardiovascular disorders, e.g., restenosis. The
term "cardiovascular disorder" or "coronary disorder" as used
interchangeably herein, refers to any disease, disorder, or state
involving the cardiovascular system, e.g., the heart, the blood
vessels, and/or the blood. A coronary disorder is generally
characterized by a narrowing of the blood vessels that supply blood
and oxygen to the heart (coronary arteries). Coronary disease may
result from the build up of fatty material and plaque. As the
coronary arteries narrow, the flow of blood to the heart can slow
or stop. Coronary disorders of the invention can apply to any
abnormality of an artery, whether structural, histological,
biochemical or any other abnormality. An example of coronary heart
disease is restenosis. In one embodiment, a coronary disorder
refers to any disease, disorder, or state involving the
cardiovascular system excluding ischemia of the heart and heart
insufficiency.
[0331] Coronary disorders in which TNF.alpha. activity is
detrimental often result from a blockage in an artery. Such a
blockage can be caused by a clot, which usually forms in a coronary
artery that has been previously narrowed from changes usually
related to atherosclerosis. For example, if the atherosclerotic
plaque inside the arterial wall cracks, it can trigger the
formation of a thrombus, or clot. Such disorders may be evidenced,
for example, by an increase in the concentration of TNF.alpha. in a
biological fluid of a subject suffering from the disorder (e.g., an
increase in the concentration of TNF.alpha. in serum, plasma,
synovial fluid, etc. of the subject), which can be detected, for
example, using an anti-TNF.alpha. antibody as described above. A
coronary disorder can be also caused by an imbalance in arterial
pressure, a malfunction of the heart, or an occlusion of a blood
vessel, e.g., by a thrombus. Coronary disorders includes both
coronary artery disease and peripheral vascular disease.
[0332] There are numerous examples of cardiac disorders in which
TNF.alpha. activity is detrimental, including restenosis. The use
of the antibodies, antibody portions, for treatment of specific
coronary disorders is discussed further below. In certain
embodiments, an antibody, antibody portion, is administered to the
subject in combination with another therapeutic agent, as described
below.
[0333] Antibodies obtained using methods of the invention may also
be used for inhibiting TNF.alpha. activity in a subject with a
cardiac disorder. The invention provides methods for inhibiting or
decreasing TNF.alpha. activity in a subject with a coronary
disorder, comprising administering to the subject an antibody, or
antibody portion, or other TNF.alpha. inhibitor of the invention
such that TNF.alpha. activity in the subject is inhibited or
decreased. Preferably, the TNF.alpha. is human TNF.alpha. and the
subject is a human subject. Alternatively, the subject can be a
mammal expressing a TNF.alpha. with which an antibody of the
invention cross-reacts. Still further the subject can be a mammal
into which has been introduced hTNF.alpha. (e.g., by administration
of hTNF.alpha. or by expression of an hTNF.alpha. transgene). An
antibody of the invention can be administered to a human subject
for therapeutic purposes.
[0334] Moreover, an antibody of the invention can be administered
to a non-human mammal expressing a TNF.alpha. with which the
antibody cross-reacts (e.g., a primate, pig or mouse) for
veterinary purposes or as an animal model of human disease.
Regarding the latter, such animal models may be useful for
evaluating the multiple-variable dose therapeutic efficacy (e.g.,
testing of dosages and time courses of administration). Commonly
used animal models for studying coronary disorders, including
restenosis, include the rat or mouse carotid artery ligation model
and the carotid artery injury model (Ferns et al. (1991) Science
253:1129; Clowes et al. (1983) Lab. Invest. 49:208; Lindner et al.
(1993) Circ Res. 73:792). In the carotid artery ligation model,
arterial blood flow is disrupted by ligation of the vessel near the
distal bifurnation. As described in Clowes et al., the carotid
artery injury model is performed such that the common carotid
artery is denuded of endothelium by the intraluminal passage of a
balloon catheter introduced through the external carotid artery. At
2 weeks, the carotid artery is markedly narrowed due to smooth
muscle cell constriction, but between 2 and 12 weeks the intimal
doubles in thickness leading to a decrease in luminal size. Any of
these models can be used to determine the potential therapeutic
action of the TNF.alpha. antibodies of the invention in the
prevention and treatment of restenosis in humans.
[0335] The invention includes treatment of cardiovascular disorders
in which TNF.alpha. activity is detrimental, wherein inhibition of
TNF.alpha. activity is expected to alleviate the symptoms and/or
progression of the coronary disease or to prevent the coronary
disease. Subjects suffering from or at risk of developing coronary
disorders can be identified through clinical symptoms. Clinical
symptoms in coronary disease often include chest pain, shortness of
breath, weakness, fainting spells, alterations in consciousness,
extremity pain, paroxysmal nocturnal dyspnea, transient ischemic
attacks and other such phenomena experienced by the patient.
Clinical signs of coronary disease can also include EKG
abnormalities, altered peripheral pulses, arterial bruits, abnormal
heart sounds, rates and wheezes, jugular venous distention,
neurological alterations and other such findings discerned by the
clinician. Coronary disorders may also be evidenced, for example,
by an increase in the concentration of TNF.alpha. in a biological
fluid of a subject suffering from the disorder (e.g., an increase
in the concentration of TNF.alpha. in serum, plasma, synovial
fluid, etc. of the subject).
[0336] Examples of a cardiovascular disorder include, but are not
limited to, coronary artery disease, angina pectoris, myocardial
infarction, cardiovascular tissue damage caused by cardiac arrest,
cardiovascular tissue damage caused by cardiac bypass, cardiogenic
shock, and hypertension, atherosclerosis, coronary artery spasm,
coronary artery disease, valvular disease, arrhythmias, and
cardiomyopathies. The use of the antibodies, antibody portions, for
treatment of specific cardiovascular diseases are discussed further
below. In certain embodiments, the antibody, antibody portion, is
administered to the subject in combination with another therapeutic
agent, as described below.
[0337] 1. Restenosis
[0338] The term "restenosis" as used herein refers to the
recurrence of stenosis, which is the narrowing or constriction of
an artery. Restenosis often occurs as a preocclusive lesion that
develops following a reconstructive procedure in a diseased blood
vessel. The term is not only applied to the recurrence of a
pre-existing stenosis, but also to previously normal vessels that
become partially occluded following vascular bypass. In another
embodiment, the invention provides a method of treating restenosis
comprising administering the antibody, or antigen binding portion
thereof, obtained using the invention to a subject who has or is at
risk of developing restenosis.
[0339] TNF.alpha. has been implicated in the pathophysiology of
restenosis (see Zhou et al. (2002) Atherosclerosis. 161:153; Javed
et al. (2002) Exp and Mol Pathol 73:104). For example, in the
murine wire carotid model, TNF -/- mice demonstrated a seven-fold
reduction in initial hyperplasia compared to wild type mice
(Zimmerman et al. (2002) Am J Phsiol Regul Integr Comp Physiol
283:R505). Restenosis can occur as the result of any type of
vascular reconstruction, whether in the coronary vasculature or in
the periphery (Colburn and Moore (1998) Myointimal Hyperplasia pp.
690-709 in Vascular Surgery: A Comprehensive Review Philadelphia:
Saunders). For example, studies have reported symptomatic
restenosis rates of 30-50% following coronary angioplasties (see
Berk and Harris (1995) Adv. Intern. Med. 40:455). After carotid
endarterectomies, as a further example, 20% of patients studied had
a luminal narrowing greater than 50% (Clagett et al. (1986) J.
Vasc. Surg. 3:10). Restenosis is evidenced in different degrees of
symptomatology which accompany preocclusive lesions in different
anatomical locations, due to a combination of factors including the
nature of the vessels involved, the extent of residual disease, and
local hemodynamics.
[0340] "Stenosis," as used herein refers to a narrowing of an
artery as seen in occlusive disorder or in restenosis. Stenosis can
be accompanied by those symptoms reflecting a decrease in blood
flow past the narrowed arterial segment, in which case the disorder
giving rise to the stenosis is termed a disease (i.e., occlusive
disease or restenosis disease). Stenosis can exist asymptomatically
in a vessel, to be detected only by a diagnostic intervention such
as an angiography or a vascular lab study.
[0341] The antibodies obtained using the method of the invention
may be used to treat a subject suffering from or at risk of
developing restenosis. A subject at risk of developing restenosis
includes a subject who has undergone PTCA. The subject may have
also had a stent inserted to prevent restenosis. The TNF.alpha.
antibody can be used alone or in combination with a stent to
prevent the re-occurrence of stenosis in a subject suffering from
cardiovascular disease.
[0342] 2. Congestive Heart Failure
[0343] TNF.alpha. has been implicated in the pathophysiology of
congestive heart failure (see Zhou et al. (2002) Atherosclerosis
161:153). Serum levels of TNF.alpha. are elevated in patients with
congestive heart failure in a manner which is directly proportional
to the severity of the disease (Levine et al. (1990) N Engl J Med
323:236; Torre-Amione et al. (1996) J Am Coll Cardiol 27:1201). In
addition, inhibitors of TNF.alpha. have also been shown to improve
congestive heart failure symptoms (Chung et al. (2003) Circulation
107:3133).
[0344] As used herein, the term "congestive heart failure" includes
a condition characterized by a diminished capacity of the heart to
supply the oxygen demands of the body. Symptoms and signs of
congestive heart failure include diminished blood flow to the
various tissues of the body, accumulation of excess blood in the
various organs, e.g., when the heart is unable to pump out the
blood returned to it by the great veins, exertional dyspnea,
fatigue, and/or peripheral edema, e.g., peripheral edema resulting
from left ventricular dysfunction. Congestive heart failure may be
acute or chronic. The manifestation of congestive heart failure
usually occurs secondary to a variety of cardiac or systemic
disorders that share a temporal or permanent loss of cardiac
function. Examples of such disorders include hypertension, coronary
artery disease, valvular disease, and cardiomyopathies, e.g.,
hypertrophic, dilative, or restrictive cardiomyopathies.
[0345] A "subject who has or is suffering from congestive heart
failure" is a subject who has a disorder involving a clinical
syndrome of diverse etiologies linked by the common denominator of
impaired heart pumping in which the heart cannot pump blood
commensurate with the requirements of the metabolizing tissues, or
can do so only from an elevated filling pressure. A "subject at
risk of developing congestive heart failure" is a subject who has a
propensity of developing congestive heart failure because of
certain factors affecting the cardiovascular system of the subject.
It is desirable to reduce the risk of or prevent the development of
congestive heart failure in these subjects. The phrase "with
congestive heart failure" includes patients who are at risk of
suffering from this condition relative to the general population,
even though they may not have suffered from it yet, by virtue of
exhibiting risk factors. For example, a patient with untreated
hypertension may not have suffered from congestive heart failure,
but is at risk because of his or her hypertensive condition. In one
embodiment of the invention, the antibody adalimumab is used to
treat a subject at risk of developing congestive heart failure.
[0346] 3. Acute Coronary Syndromes
[0347] TNF.alpha. has been implicated in the pathophysiology of
acute coronary syndromes (see Libby (1995) Circulation 91:2844).
Acute coronary syndromes include those disorders wherein the
subject experiences pain due to a blood flow restriction resulting
in not enough oxygen reaching the heart. Studies have found that
TNF.alpha. plays a role in acute coronary syndromes. For example,
in a novel rat heterotropic cardiac transplantation-coronary
ligation model capable of inducing myocardial infarction in the
absence of downstream hemodynamic effects, administration of
chimeric soluble TNF receptor (sTNFR) abolished transient LV
remodeling and dysfunction (Nakamura, et al. (2003) J. Cardiol.
41:41). It was also found that direct injection of an sTNFR
expression plasmid to the myocardium, resulted in a reduction in
the infarction size in acute myocardial infarction (AMI)
experimental rats (Sugano et al. (2002) FASEB J 16:1421).
[0348] In one embodiment, a TNF.alpha. antibody is used for the
treatment or prevention of an acute coronary syndrome in a subject,
wherein the acute coronary syndrome is a myocardial infarction or
angina.
[0349] As used herein, the term "myocardial infarction" or "MI"
refers to a heart attack. A myocardial infarction involves the
necrosis or permanent damage of a region of the heart due to an
inadequate supply of oxygen to that area. This necrosis is
typically caused by an obstruction in a coronary artery from either
atherosclerosis or an embolis. MIs which are treated by the
TNF.alpha. antibody obtained using the methods of the invention
include both Q-wave and non-Q-wave myocardial infarction. Most
heart attacks are caused by a clot that blocks one of the coronary
arteries (the blood vessels that bring blood and oxygen to the
heart muscle). For example, a clot in the coronary artery
interrupts the flow of blood and oxygen to the heart muscle,
leading to the death of heart cells in that area. The damaged heart
muscle permanently loses its ability to contract, and the remaining
heart muscle needs to compensate for it. An MI can also be caused
by overwhelming stress in the individual.
[0350] The term "angina" refers to spasmodic, choking, or
suffocative pain, and especially as denoting angina pectoris which
is a paroxysmal thoracic pain due, most often, to anoxia of the
myocardium. Angina includes both variant angina and exertional
angina. A subject having angina has ischemic heart disease which is
manifested by sudden, severe, pressing substemal pain that often
radiates to the left shoulder and along the left arm. TNF.alpha.
has been implicated in angina, as TNF.alpha. levels are upregulated
in patients with both MI and stable angina (Balbay et al. (2001)
Angiology 52109).
[0351] 4. Artherosclerosis
[0352] "Atherosclerosis" as used herein refers to a condition in
which fatty material is deposited along the walls of arteries. This
fatty material thickens, hardens, and may eventually block the
arteries. Atherosclerosis is also referred to arteriosclerosis,
hardening of the arteries, and arterial plaque buildup. Polyclonal
antibodies directed against TNF.alpha. have been shown to be
effective at neutralizing TNF.alpha. activity resulting in
inflammation and restenosis in the rabbit atherosclerotic model
(Zhou et al., supra). Accordingly, a TNF.alpha..quadrature.antibody
may be used to treat or prevent subjects afflicted with or at risk
of having atherosclerosis.
[0353] 5. Cardiomyopathy
[0354] The term "cardiomyopathy" as used herein is used to define
diseases of the myocardium wherein the heart muscle or myocardium
is weakened, usually resulting in inadequate heart pumping.
Cardiomyopathy can be caused by viral infections, heart attacks,
alcoholism, long-term, severe hypertension (high blood pressure),
or by autoimmune causes.
[0355] In approximately 75-80% of heart failure patients coronary
artery disease is the underlying cause of the cardiomyopathy and is
designated "ischemic cardiomyopathy." Ischemic cardiomyopathy is
caused by heart attacks, which leave scars in the heart muscle or
myocardium. The affected myocardium is then unable to contribute to
the heart pumping function. The larger the scars or the more
numerous the heart attacks, the higher the chance there is of
developing ischemic cardiomyopathy.
[0356] Cardiomyopathies that are not attributed to underlying
coronary artery disease, and are designated "non-ischemic
cardiomyopathies." Non-ischemic cardiomyopathies include, but are
not limited to idiopathic cardiomyopathy, hypertrophic
cardiomyopathy, alcoholic cardiomyopathy, dilated cardiomyopathy,
peripartum cardiomyopathy, and restrictive cardiomyopathy.
I. Spondyloarthropathies
[0357] TNF.alpha. has been implicated in the pathophysiology of a
wide variety of disorders, including inflammatory diseases such as
spondyloarthopathies (see e.g., Moeller et al. (1990) Cytokine
2:162; U.S. Pat. No. 5,231,024; European Patent Publication No. 260
610). The invention provides multiple-variable dose methods for
inhibiting TNF.alpha. activity in a subject suffering from a
spondyloarthropathy, which method comprises administering to the
subject an antibody, antibody portion, such that TNF.alpha.
activity in the subject suffering from a spondyloarthropathy is
inhibited.
[0358] As used herein, the term "spondyloarthropathy" or
"spondyloarthropathies" is used to refer to any one of several
diseases affecting the joints of the spine, wherein such diseases
share common clinical, radiological, and histological features. A
number of spondyloarthropathies share genetic characteristics, i.e.
they are associated with the HLA-B27 allele. In one embodiment, the
term spondyloarthropathy is used to refer to any one of several
diseases affecting the joints of the spine, excluding ankylosing
spondylitis, wherein such diseases share common clinical,
radiological, and histological features. Examples of
spondyloarthropathies include ankylosing spondylitis, psoriatic
arthritis/spondylitis, enteropathic arthritis, reactive arthritis
or Reiter's syndrome, and undifferentiated spondyloarthropathies.
Examples of animal models used to study spondyloarthropathies
include ank/ank transgenic mice, HLA-B27 transgenic rats (see
Taurog et al. (1998) The Spondylarthritides. Oxford:Oxford
University Press).
[0359] The multiple-variable dose methods of the invention can also
be used to treat subjects who are at risk of developing a
spondyloarthropathy using multiple-variable dose methods. Examples
of subjects who are at risk of having spondyloarthropathies include
humans suffering from arthritis. Spondyloarthropathies can be
associated with other forms of arthritis, including rheumatoid
arthritis. In one embodiment of the invention, antibodies are used
in multiple-variable dose methods to treat a subject who suffers
from a spondyloarthropathy associated with rheumatoid arthritis.
Examples of spondyloarthropathies which can be treated with a
TNF.alpha. antibody are described below:
[0360] 1. Ankylosing Spondylitis (AS)
[0361] Tumor necrosis factor has been implicated in the
pathophysiology of ankylosing spondylitis (see Verjans et al.
(1991) Arthritis Rheum. 34:486; Verjans et al. (1994) Clin Exp
Immunol. 97:45; Kaijtzel et al. (1999) Hum Immunol. 60:140).
Ankylosing spondylitis (AS) is an inflammatory disorder involving
inflammation of one or more vertebrae. AS is a chronic inflammatory
disease that affects the axial skeleton and/or peripheral joints,
including joints between the vertebrae of the spine and sacroiliac
joints and the joints between the spine and the pelvis. AS can
eventually cause the affected vertebrae to fuse or grow together.
Spondyarthropathies, including AS, can be associated with psoriatic
arthritis (PsA) and/or inflammatory bowel disease (IBD), including
ulcerative colitis and Crohn's disease.
[0362] Early manifestations of AS can be determined by radiographic
tests, including CT scans and MRI scans. Early manifestations of AS
often include scroiliitis and changes in the sacroiliac joints as
evidenced by the blurring of the cortical margins of the
subchrondral bone, followed by erosions and sclerosis. Fatigue has
also been noted as a common symptom of AS (Duffy et al. (2002) ACR
66th Annual Scientific Meeting Abstract). Accordingly,
multiple-variable dose methods comprising administering an
antibody, or antigen-binding fragment thereof, of the invention can
be used to treat AS.
[0363] In one embodiment, the multiple-variable dose method of the
invention is used to treat a spondyloarthropathy associated with
IBD, including AS. AS is often treated with nonsteroidal
anti-inflammatory medications (NSAIDs), such as aspirin or
indomethacin. Accordingly, a TNF.alpha. antibody used in the
multiple-variable dose method of the invention may also be
administered in combination with agents commonly used to reduce
inflammation and pain commonly associated with ankylosing
spondylitis.
[0364] 2. Psoriatic Arthritis
[0365] Tumor necrosis factor has been implicated in the
pathophysiology of psoriatic arthritis (PsA) (Partsch et al. (1998)
Ann Rheum Dis. 57:691; Ritchlin et al. (1998) J. Rheumatol.
25:1544). As referred to herein, psoriatic arthritis or psoriasis
associated with the skin, refers to chronic inflammatory arthritis
which is associated with psoriasis, which is a common chronic skin
condition that causes red patches on the body. About 1 in 20
individuals with psoriasis will develop arthritis along with the
skin condition, and in about 75% of cases, psoriasis precedes the
arthritis. PsA exhibits itself in a variety of ways, ranging from
mild to severe arthritis, wherein the arthritis usually affects the
fingers and the spine. When the spine is affected, the symptoms are
similar to those of ankylosing spondylitis, as described above. The
TNF.alpha. antibody, or antigen-binding fragment thereof, obtained
using the invention can be used for treatment of PsA.
[0366] PsA is sometimes associated with arthritis mutilans.
Arthritis mutilans refers to a disorder which is characterized by
excessive bone erosion resulting in a gross, erosive deformity
which mutilates the joint. In one embodiment, antibodies obtained
using the method of the invention are used to treat arthritis
mutilans.
[0367] 3. Reactive Arthritis/Reiter's Syndrome
[0368] Tumor necrosis factor has been implicated in the
pathophysiology of reactive arthritis, which is also referred to as
Reiter's syndrome (Braun et al. (1999) Arthritis Rheum.
42(10):2039). Reactive arthritis (ReA) refers to arthritis which
complicates an infection elsewhere in the body, often following
enteric or urogenital infections. ReA is often characterized by
certain clinical symptoms, including inflammation of the joints
(arthritis), urethritis, conjunctivitis, and lesions of the skin
and mucous membranes. In addition, ReA can occurs following
infection with a sexually transmitted disease or dysenteric
infection, including chlamydia, campylobacter, salmonella, or
yersinia. Accordingly, antibodies obtained using the method of the
invention may be used to treat ReA.
[0369] 4. Undifferentiated Spondyloarthropathies
[0370] In one embodiment, antibodies obtained using methods of the
invention are used to treat subjects suffering from
undifferentiated spondyloarthropathies (see Zeidler et al. (1992)
Rheum Dis Clin North Am. 18:187). Other terms used to describe
undifferentiated spondyloarthropathies include seronegative
oligoarthritis and undifferentiated oligoarthritis.
Undifferentiated spondyloarthropathies, as used herein, refers to a
disorder wherein the subject demonstrates only some of the symptoms
associated with a spondyloarthropathy. This condition is usually
observed in young adults who do not have IBD, psoriasis, or the
classic symptoms of AS or Reiter's syndrome. In some instances,
undifferentiated spondyloarthropathies may be an early indication
of AS. In one embodiment, the invention comprises administering a
TNF.alpha. antibody, or antigen-binding fragment thereof, obtained
using the claimed process to treat undifferentiated
spondyloarthropathies.
J. Metabolic Disorders
[0371] TNF.alpha. has been implicated in the pathophysiology of a
wide variety of disorders, including metabolic disorders, such as
diabetes and obesity (Spiegelman and Hotamisligil (1993) Cell
73:625; Chu et al. (2000) Int J Obes Relat Metab Disord. 24:1085;
Ishii et al. (2000) Metabolism. 49:1616). The term "metabolic
disorder," as used herein, refers to diseases or disorders which
affect how the body processes substances needed to carry out
physiological functions. Examples of metabolic disorders include,
but are not limited to, diabetes and obesity. In one embodiment of
the invention, the term "metabolic disorder" is used to refer to
disorders which affect how the body processes substances needed to
carry out physiological functions, excluding autoimmune
diabetes.
[0372] The invention provides methods for inhibiting TNF.alpha.
activity in a subject suffering from such a metabolic disorder,
which method comprises administering to the subject an antibody,
antibody portion, such that TNF.alpha. activity in the subject
suffering from a metabolic disorder is inhibited. TNF.alpha.
antibodies can also be used to treat subjects who are at risk of
developing a metabolic disorder.
[0373] Metabolic disorders are often associated with arthritis,
including rheumatoid arthritis. In one embodiment, a TNF.alpha.
inhibitor, such as an antibody, is used in a multiple-variable dose
regimen in a subject who suffers from a metabolic disorder
associated with rheumatoid arthritis. In another embodiment, the
invention comprises administering a TNF.alpha. antibody to treat
disorders associated with diabetes or obesity.
[0374] Examples of animal models for evaluating the efficacy of a
TNF.alpha. antibody for the treatment of a metabolic disorder
include NOD transgenic mice, Akita mice, NSY transgenic mice and
ob/ob mice (see Baeder et al. (1992) Clin Exp Immunol. 89:174;
Haseyama et al. (2002) Tohoku J Exp Med. 198:233; Makino et al.
(1980): Exp. Anim. 29:1; Kolb (1987) Diabetes/Metabolism Reviews
3:751; Hamada et al. (2001) Metabolism. 50:1282; Coleman, (1978)
Diabetologia, 14:141; Bailey et al. (1982) Int. J. Obesity 6:11).
Examples of animal models used to study vasculitis includes the
mouse HSV model (Behcet's disease), the mouse L. casei model
(Kawasaki's disease), and the mouse ANCA model (Kawasaki's
disease). Other models of vasculitis include the MCH5-lpr/lpr
strain (Nose et al. (1996) Am. J. Path. 149:1763) and the SCG/Kj
strain of mice (Kinjoh et al. (1993) Proc. Natl. Acad. Sci., USA
90:3413). These mice strains spontaneously develop crescentic
glomerulonephritis and necrotizing vasculitis of the small arteries
and arterioles of the spleen, stomach, heart, uterus and ovaries.
These animals develop hypergammaglobulinemia and ANCA
autoantibodies that react with myeloperoxidase (MPO). Additionally,
immunization of rats with human MPO results in ANCA-associated
necrotizing crescentic glomerulonephritis (Brouwer et al. (1993) J.
Exp. Med. 177:905).
[0375] Metabolic disorders affect how the body processes substances
needed to carry out physiological functions. A number of metabolic
disorders of the invention share certain characteristics, i.e. they
are associated the insulin resistance, lack of ability to regulate
blood sugar, weight gain, and increase in body mass index. Examples
of metabolic disorders include diabetes and obesity. Examples of
diabetes include type 1 diabetes mellitus, type 2 diabetes
mellitus, diabetic neuropathy, peripheral neuropathy, diabetic
retinopathy, diabetic ulcerations, retinopathy ulcerations,
diabetic macrovasculopathy, and obesity. Examples of metabolic
disorders which can be treated using multiple-variable dose methods
comprising administration of a TNF.alpha. antibody are described in
more detail below:
[0376] 1. Diabetes
[0377] Tumor necrosis factor has been implicated in the
pathophysiology of diabetes. (see e.g., Navarro et al. (2003) Am J
Kidney Dis. 42:53; Daimon et al. (2003) Diabetes Care. 26:2015;
Zhang et al. (1999) J Tongji Med. Univ. 19:203; Barbieri et al.
(2003) Am J. Hypertens. 16:537) For example, TNF.alpha. is
implicated in the pathophysiology for insulin resistance. It has
been found that serum TNF levels in patients with gastrointestinal
cancer correlates with insulin resistance (see e.g., McCall et al.
(1992) Br. J. Surg. 79:1361).
[0378] The term "diabetes" or "diabetic disorder" or "diabetes
mellitus," as used interchangeably herein, refers to a disease
which is marked by elevated levels of sugar (glucose) in the blood.
Diabetes can be caused by too little insulin (a chemical produced
by the pancreas to regulate blood sugar), resistance to insulin, or
both. Diabetes includes the two most common types of the disorder,
namely type I diabetes and type II diabetes, which both result from
the body's inability to regulate insulin. Insulin is a hormone
released by the pancreas in response to increased levels of blood
sugar (glucose) in the blood.
[0379] The term "type 1 diabetes," as used herein, refers to a
chronic disease that occurs when the pancreas produces too little
insulin to regulate blood sugar levels appropriately. Type 1
diabetes is also referred to as insulin-dependent diabetes
mellitus, IDMM, juvenile onset diabetes, and diabetes--type I. Type
1 diabetes represents is the result of a progressive autoimmune
destruction of the pancreatic .beta.-cells with subsequent insulin
deficiency.
[0380] The term "type 2 diabetes," refers to a chronic disease that
occurs when the pancreas does not make enough insulin to keep blood
glucose levels normal, often because the body does not respond well
to the insulin. Type 2 diabetes is also referred to as
noninsulin-dependent diabetes mellitus, NDDM, and diabetes--type
II
[0381] Diabetes is can be diagnosed by the administration of a
glucose tolerance test. Clinically, diabetes is often divided into
several basic categories. Primary examples of these categories
include, autoimmune diabetes mellitus, non-insulin-dependent
diabetes mellitus (type 1 NDDM), insulin-dependant diabetes
mellitus (type 2 IDDM), non-autoimmune diabetes mellitus,
non-insulin-dependant diabetes mellitus (type 2 NIDDM), and
maturity-onset diabetes of the young (MODY). A further category,
often referred to as secondary, refers to diabetes brought about by
some identifiable condition which causes or allows a diabetic
syndrome to develop. Examples of secondary categories include,
diabetes caused by pancreatic disease, hormonal abnormalities,
drug- or chemical-induced diabetes, diabetes caused by insulin
receptor abnormalities, diabetes associated with genetic syndromes,
and diabetes of other causes. (see e.g., Harrison's (1996)
14.sup.th ed., New York, McGraw-Hill).
[0382] Diabetes is often treated with diet, insulin dosages, and
various medications described herein. Accordingly, a TNF.alpha.
antibody may also be administered in combination with agents
commonly used to treat metabolic disorders and pain commonly
associated with diabetes.
[0383] In addition, the phrase "disorders associated with
diabetes," as used herein, refers to conditions and other diseases
which are commonly associated with or related to diabetes. Example
of disorders associated with diabetes include, for example,
hyperglycemia, hyperinsulinaemia, hyperlipidaemia, insulin
resistance, impaired glucose metabolism, obesity, diabetic
retinopathy, macular degeneration, cataracts, diabetic nephropathy,
glomerulosclerosis, diabetic neuropathy, erectile dysfunction,
premenstrual syndrome, vascular restenosis, ulcerative colitis,
coronary heart disease, hypertension, angina pectoris, myocardial
infarction, stroke, skin and connective tissue disorders, foot
ulcerations, metabolic acidosis, arthritis, and osteoporosis.
[0384] Diabetes manifests itself in the foregoing categories and
can cause several complications that are discussed in the following
sections. Accordingly, the antibody, or antigen-binding fragment
thereof, of the invention can be used to treat diabetes. In one
embodiment, a TNF.alpha. antibody, or antigen-binding fragment
thereof, is used to treat diabetes associated with the above
identified categories. In another embodiment, the invention
includes administering a TNF.alpha. antibody to treat disorders
associated with diabetes. Diabetes manifests itself in many
complications and conditions associated with diabetes, including
the following categories:
[0385] a. Diabetic Neuropathy and Peripheral Neuropathy
[0386] Tumor necrosis factor has been implicated in the
pathophysiology of diabetic neuropathy and peripheral neuropathy.
(See Benjafield et al. (2001) Diabetes Care. 24:753; Qiang et al.
(1998) Diabetologia. 41:1321; Pfeiffer et al. (1997) Horm Metab
Res. 29:111).
[0387] The term "neuropathy," also referred to as nerve
damage-diabetic, as used herein, refers to a common complication of
diabetes in which nerves are damaged as a result of hyperglycemia
(high blood sugar levels). A variety of diabetic neuropathies are
recognized, such as distal sensorimotror polyneuropathy, focal
motor neuropathy, and autonomic neuropathy.
[0388] The term "peripheral neuropathy," also known as peripheral
neuritis and diabetic neuropathy, as used herein, refers to the
failure of the nerves to carry information to and from the brain
and spinal cord. Peripheral neuropathy produces symptoms such as
pain, loss of sensation, and the inability to control muscles. In
some cases, the failure of nerves to control blood vessels,
intestinal function, and other organs results in abnormal blood
pressure, digestion, and loss of other basic involuntary processes.
Peripheral neuropathy may involve damage to a single nerve or nerve
group (mononeuropathy) or may affect multiple nerves
(polyneuropathy).
[0389] Neuropathies that affect small myelinated and unmyelinated
fibers of the sympathetic and parasympathetic nerves are known as
"peripheral neuropathies." Furthermore, the related disorder of
peripheral neuropathy, also known as peripheral neuritis and
diabetic neuropathy, refers to the failure of the nerves to carry
information to and from the brain and spinal cord. This produces
symptoms such as pain, loss of sensation, and the inability to
control muscles. In some cases, failure of nerves controlling blood
vessels, intestinal function, and other organs results in abnormal
blood pressure, digestion, and loss of other basic involuntary
processes. Peripheral neuropathy may involve damage to a single
nerve or nerve group (mononeuropathy) or may affect multiple nerves
(polyneuropathy).
[0390] The term "diabetic neuropathy" refers to a common
complication of diabetes in which nerves are damaged as a result of
hyperglycemia (high blood sugar levels). Diabetic neuropathy is
also referred to as neuropathy and nerve damage-diabetic. A variety
of diabetic neuropathies are recognized, such as distal
sensorimotror polyneuropathy, focal motor neuropathy, and autonomic
neuropathy.
[0391] b. Diabetic Retinopathy
[0392] Tumor necrosis factor has been implicated in the
pathophysiology of diabetic retinopthy (Scholz et al. (2003) Trends
Microbiol. 11:171). The term "diabetic retinopathy" as used herein,
refers to progressive damage to the eye's retina caused by
long-term diabetes. Diabetic retinopathy, includes proliferative
retinopathy. Proliferative neuropathy in turn includes includes
neovascularization, pertinal hemmorrhave and retinal
detachment.
[0393] In advanced retinopathy, small vessels proliferate on the
surface of the retina. These blood vessels are fragile, tend to
bleed and can cause peretinal hemorrhages. The hemorrhage can
obscure vision, and as the hemorrhage is resorbed fibrous tissue
forms predisposing to retinal detachments and loss of vision. In
addition, diabetic retinopathy includes proliferative retinopathy
which includes neovascularization, pertinal hemmorrhave and retinal
detachment. Diabetic retinopathy also includes "background
retinopathy" which involves changes occurring with the layers of
the retina.
[0394] c. Diabetic Ulcerations and Retinopathy Ulcerations
[0395] Tumor necrosis factor has been implicated in the
pathophysiology of diabetic ulcerations, (see Lee et al. (2003) Hum
Immunol. 64:614; Navarro et al. (2003) Am J Kidney Dis. 42:53;
Daimon et al (2003) Diabetes Care. 26:2015; Zhang et al. (1999) J
Tongji Med. Univ. 19:203; Barbieri et al. (2003) Am J. Hypertens.
16:537; Venn et al. (1993) Arthritis Rheum. 36:819; Westacott et
al. (1994) J. Rheumatol. 21:1710).
[0396] The term "diabetic ulcerations," as used herein, refers to
an ulcer which results as a complication of diabetes. An ulcer is a
crater-like lesion on the skin or mucous membrane caused by an
inflammatory, infectious, malignant condition, or metabolic
disorder. Typically diabetic ulcers can be found on limbs and
extremities, more typically the feet. These ulcers, caused by
diabetic conditions, such as neuropathy and a vascular
insufficiency, can lead to ischemia and poor wound healing. More
extensive ulcerations may progress to osteomyelitis. Once
osteomyelitis develops, it may be difficult to eradicate with
antibiotics alone and amputation maybe necessary.
[0397] The term "retinopathy ulcerations," as used herein refers to
an ulcer which causes or results in damages to the eye and the
eye's retina. Retinopathy ulcerations may include conditions such
has retinoathic hemmorages.
[0398] d. Diabetic Macrovasculopathy
[0399] Tumor necrosis factor has been implicated in the
pathophysiology of diabetic macrovasculopathy (Devaraj et al.
(2000) Circulation. 102:191; Hattori et al. (2000) Cardiovasc Res.
46:188; Clausell et al. (1999) Cardiovasc Pathol. 8:145). The term
"diabetic macrovasculopathy," also referred to as "macrovascular
disease," as used herein, refers to a disease of the blood vessels
that results from diabetes. Diabetic macrovasculopathy complication
occurs when, for example, fat and blood clots build up in the large
blood vessels and stick to the vessel walls. Diabetic
macrovasculopathies include diseases such as coronary disease,
cerebrovascular disease, and peripheral vascular disease,
hyperglycaemia and cardiovascular disease, and strokes.
[0400] 2. Obesity
[0401] Tumor necrosis factor has been implicated in the
pathophysiology of obesity (see e.g., Pihlajamaki J et al. (2003)
Obes Res. 11:912; Barbieri et al. (2003) Am J Hypertens. 16:537;
Tsuda et al. (2003) J. Nutr. 133:2125). The term "obesity" as used
herein, refers to a condition in which the subject has an excess of
body fat relative to lean body mass. In one embodiment, obesity
refers to a condition in which an individual weighs at least about
20% or more over the maximum desirable for their height. When an
adult is more than 100 pounds overweight, he or she is considered
to be "morbidly obese." In another embodiment, obesity is defined
as a BMI (body mass index) over 30 kg/m2. Obesity increases a
person's risk of illness and death due to diabetes, stroke,
coronary artery disease, hypertension, high cholesterol, and kidney
and gallbladder disorders. Obesity may also increase the risk for
some types of cancer, and may be a risk factor for the development
of osteoarthritis and sleep apnea.
K. Anemia
[0402] TNF.alpha. has been implicated in the pathophysiology of a
wide variety of anemias (see e.g., Jongen-Lavrencic et al. (1997)
J. Rheumatol. 24:1504; Demeter et al. (2002) Ann Hematol. 81:566;
DiCato (2003) The Oncologist 8 (suppl 1):19). The invention
provides a method for inhibiting TNF.alpha. activity in a subject
suffering from anemia, which method comprises administering to the
subject an antibody, antibody portion, such that TNF.alpha.
activity in the subject suffering from anemia is inhibited. In one
embodiment, the anemia is associated with rheumatoid arthritis.
[0403] The term "anemia" as used herein, refers to an abnormally
low number of circulating red cells or a decreased concentration of
hemoglobin in the blood. Examples of anemia related to rheumatoid
arthritis include, for example, anemia of chronic disease, iron
deficiency anemia, and autoimmune hemolytic anemia. In one
embodiment, the invention provides a method of treating anemias
related to, for example, anemias related to rheumatoid arthritis,
anemias of infection and chronic inflammatory diseases, iron
deficiency anemia, autoimmune hemolytic anemia, myelophthisic
anemia, aplastic anemia, hypoplastic anemia, pure red cell aplasia
and anemia associated with renal failure or endocrine disorders,
megaloblastic anemias, defects in heme or globin synthesis, anemia
caused by a structural defect in red blood cells, e.g., sickle-cell
anemia, and anemias of unknown origins such as sideroblastic
anemia, anemia associated with chronic infections such as malaria,
trypanosomiasis, HIV, hepatitis virus or other viruses, and
myelophthisic anemias caused by marrow deficiencies.
[0404] Examples of animal models used to study anemia include rats
inoculated with peptidolglycan-polysaccharide polymers (see Coccia
et al., (2001) Exp Hematology. 29:1201-1209). Examples of animal
models used to study pain are well known in the art, and include
the rat sciatic nerve ligation model, and the rat segmental spinal
nerve ligation model (see Bennett and Zie, (1988) Pain. 33:87-107;
Kim and Chung, (1992) Pain 50:355-363).
L. Pain
[0405] TNF.alpha. has been implicated in the pathophysiology of a
wide variety of pain syndromes (see e.g., Sorkin et al. (1997)
Neuroscience. 81:255; Huygen et al. (2002) Mediators Inflamm.
11:47; Parada et al. (2003) Eur J. Neurosci. 17:1847). The term
"pain" as used herein, refers to all types of pain. The term shall
refer to acute and chronic pains, such as neuropathic pain and
post-operative pain, chronic lower back pain, cluster headaches,
herpes neuralgia, phantom limb pain, central pain, dental pain,
opioid-resistant pain, visceral pain, surgical pain, bone injury
pain, pain during labor and delivery, pain resulting from burns,
including sunburn, post partum pain, migraine, angina pain, and
genitourinary tract-related pain including cystitis. The term also
includes nociceptive pain or nociception.
[0406] The invention provides methods for inhibiting TNF.alpha.
activity in a subject suffering from such a pain disorder, which
method comprises administering to the subject an antibody, antibody
portion, such that TNF.alpha. activity in the subject suffering
from pain is inhibited. Pain has been defined in a variety of ways,
including nociceptive pain and neuropathic pain. The most commonly
experienced form of pain may be defined as the effect of a stimulus
on nerve endings, which results in the transmission of impulses to
the cerebrum. Pain is also commonly associated with inflammatory
disorders, including, for example, rheumatoid arthritis. In one
embodiment, the antibody of the invention is used to treat a
subject who suffers from pain associated with rheumatoid arthritis.
Examples of pain disorders in which TNF.alpha. activity is
detrimental are discussed further below.
[0407] 1. Neuropathic Pain
[0408] Tumor necrosis factor has been implicated in the
pathophysiology of neuropathic pain (see Sommer (1999) Schmerz.
13:315; Empl et al., (2001) Neurology. 56:1371; Schafers et al.
(2003) J. Neurosci. 23:3028). As used herein the term "neuropathic
pain" refers to pain that results from injury to a nerve, spinal
cord, or brain, and often involves neural supersensitivity.
Examples of neuropathic pain include chronic lower back pain, pain
associated with arthritis, cancer-associated pain, herpes
neuralgia, phantom limb pain, central pain, opioid resistant
neuropathic pain, bone injury pain, and pain during labor and
delivery. Other examples of neuropathic pain include post-operative
pain, cluster headaches, dental pain, surgical pain, pain resulting
from severe, for example third degree, burns, post partum pain,
angina pain, genitourinary tract related pain, and including
cystitis.
[0409] Neuropathic pain is distinguished from nociceptive pain.
Pain involving a nociceptive mechanism usually is limited in
duration to the period of tissue repair and generally is alleviated
by available analgesic agents or opioids (Myers (1995) Regional
Anesthesia 20:173). Neuropathic pain typically is long-lasting or
chronic and often develops days or months following an initial
acute tissue injury. Neuropathic pain can involve persistent,
spontaneous pain as well as allodynia, which is a painful response
to a stimulus that normally is not painful. Neuropathic pain also
can be characterized by hyperalgesia, in which there is an
accentuated response to a painful stimulus that usually is trivial,
such as a pin prick. Unlike nociceptive pain, neuropathic pain
generally is resistant to opioid therapy (Myers, supra, 1995).
Accordingly, antibodies obtained using methods of the invention can
be used to treat neuropathic pain.
[0410] 2. Nociceptive Pain
[0411] As used herein the term "nociceptive pain" refers to pain
that is transmitted across intact neuronal pathways, i.e., pain
caused by injury to the body. Nociceptive pain includes somatic
sensation and normal function of pain, and informs the subject of
impending tissue damage. The nociceptive pathway exists for
protection of the subject, e.g., the pain experienced in response
to a burn). Nociceptive pain includes bone pain, visceral pain, and
pain associated with soft tissue.
[0412] Tumor necrosis factor has been implicated in the
pathophysiology of visceral pain (see Coelho et al. (2000) Am J
Physiol Gastrointest Liver Physiol. 279:G781; Coelho et al. (2000)
Brain Res Bull. 52:223). Visceral pain is used to refer to
nociceptive pain that is mediated by receptors on A-delta and C
nerve fibers. A-delta and C-nerve fibers are which are located in
skin, bone, connective tissue, muscle and viscera. Visceral pain
can be vague in distribution, spasmodic in nature and is usually
described as deep, aching, squeezing and colicky in nature.
Examples of visceral pain include pain associated with a heart
attack, wherein the visceral pain can be felt in the arm, neck
and/or back, and liver capsule pain, wherein the visceral pain can
be felt in the back and/or right shoulder. Accordingly, antibodies
obtained using the invention can be used to treat visceral
pain.
[0413] M. Hepatic Disorders
[0414] TNF.alpha. has been implicated in the pathophysiology of a
wide variety of hepatic disorders (see e.g., Colletti et al. (1990)
J Clin Invest. 85:1936; Tiegs (1997) Acta Gastroenterol Belg.
60:176; Fernandez et al. (2000) J Endotoxin Res. 6:321). The
invention provides methods for inhibiting TNF.alpha. activity in a
subject suffering from such a hepatic disorder.
[0415] As used herein, the term "a hepatic disorder in which
TNF.alpha. activity is detrimental" is intended to include diseases
and other disorders of the liver or conditions associated with
hepatocellular injury or a biliary tract disorders in which the
presence of TNF.alpha. in a subject suffering from the disorder has
been shown to be or is suspected of being either responsible for
the pathophysiology of the disorder or a factor that contributes to
a worsening of the disorder. Accordingly, a hepatic disorder in
which TNF.alpha. activity is detrimental is a disorder in which
inhibition of TNF.alpha. activity is expected to alleviate the
symptoms and/or progression of the hepatic disorder. In one
embodiment, hepatic disorders refers to a human liver disease or
condition associated with hepatocellular injury or a biliary tract
disorder excluding hepatitis, alcoholic hepatitis, and viral
hepatitis.
[0416] Examples of animal models used for evaluating the
therapeutic efficacy of an agent for treating a hepatic disorder
using multiple-variable dose methods include the chimpanzee
hepatitis C virus model (see Shimizu et al. (1990) Proc Natl Acad.
Sci. USA 87:6441). Examples of animal models used to study skin and
nail disorder disorders include, for example, the severe combined
immunodeficient (SCID) mouse model (psoriasis) and the Smith line
(SL) chicken and depigmenting mouse (vitiligo) (see Nickoloff
(2000) Investig Dermatol Symp Proc. 5:67; Austin et al. (1995) Am
J. Pathol. 146:1529; Lerner et al. (1986) J Invest Dermatol.
87:299).
[0417] Hepatic disorders include many diseases and disorders
wherein the liver functions improperly or ceases to function.
Hepatocellular injuries can include alcoholic cirrhosis, .alpha.1
antitypsin deficiency, autoimmune cirrhosis, cryptogenic cirrhosis,
fulminant hepatitis, hepatitis B and C, and steatohepatitis.
Examples of biliary tract disorders include cystic fibrosis,
primary biliary cirrhosis, sclerosing cholangitis and biliary
obstruction (Wiesner (1996) "Current Indications, Contra
Indications and Timing for Liver Transplantation" in
Transplantation of the Liver, Saunders (publ.); Busuttil and
Klintmalm (eds.) Chapter 6; Klein (1998) Partial Hypertension: The
Role of Liver Transplantation, Musby (publ.) in Current Surgical
Therapy 6.sup.th Ed. Cameron, J. (ed).
[0418] The term "hepatitis" refers to inflammation of the liver.
Hepatitis can be caused by infections with various organisms,
including bacteria, viruses (Hepatitis A, B, C, etc.), or
parasites. Chemical toxins such as alcohol, drugs, or poisonous
mushrooms can also damage the liver and cause it to become
inflamed. A rare but extremely dangerous cause of hepatitis results
from overdose of acetaminophen (Tylenol), which can be deadly. In
addition, immune cells in the body may attack the liver and cause
autoimmune hepatitis. Hepatitis may resolve quickly (acute
hepatitis), or cause long-term disease (chronic hepatitis). In some
instances, progressive liver damage or liver failure may result.
The incidence and severity of hepatitis vary depending on many
factors, including the cause of the liver damage and any underlying
illnesses in a patient.
[0419] In one embodiment, the invention features methods for
treating a hepatic disorder in which TNF.alpha. activity is
detrimental, comprising administering to a subject an effective
amount of a TNF.alpha. inhibitor in an induction dose and
subsequently in a treatment dose, such that said disorder is
treated. In one embodiment, the hepatic disorder is selected from
the group consisting of hepatitis C virus, autoimmune hepatitis,
fatty-liver disease, hepatitis B virus, hepatotoxicity, and
non-alcoholic hepatitis, including non-alcoholic steatohepatitis
(NASH). Examples of hepatic disorders are further described
below.
[0420] 1. Hepatitis C Virus (HCV)
[0421] Tumor necrosis factor has been implicated in the
pathophysiology of the hepatitis C virus (see Gonzalez-Amaro.
(1994) J Exp Med. 179:841; Nelson et al. (1997) Dig Dis Sci
42:2487; Kallinowski et al. (1998) Clin Exp Immunol. 111:269). The
term "hepatitis C virus" or "HCV" is used to describe the hepatitis
virus which is the causative agent of non-A, non-B hepatitis.
Hepatitis C virus causes an inflammation of the liver. HCV
infection causes hepatitis C. Hepatitis C in the acute stage is, in
general, milder than hepatitis B, but a greater proportion of such
infections become chronic. HCV is a major cause of acute hepatitis
and chronic liver disease, including cirrhosis and liver cancer.
HCV is one of the viruses (A, B, C, D, and E), which together
account for the vast majority of cases of viral hepatitis. It is an
enveloped RNA virus in the flaviviridae family which appears to
have a narrow host range. An important feature of the virus is the
relative mutability of its genome, which in turn is probably
related to the high propensity (80%) of inducing chronic infection.
HCV is clustered into several distinct genotypes which may be
important in determining the severity of the disease and the
response to treatment. In one embodiment, the invention provides a
multiple-variable dose method for treating HCV.
[0422] 2. Autoimmune Hepatitis (AIH)
[0423] Tumor necrosis factor has been implicated in the
pathophysiology of autoimmune hepatitis (see Cookson et al., (1999)
Hepatology 30:851; Jazrawi et al., (2003) Liver Transpl. 9:377). As
used herein, "autoimmune hepatitis" refers to a hepatic disorder
characterized by inflammation of the liver caused by rogue immune
cells that mistake the liver's normal cells for a foreign tissue or
pathogen (disease-causing agent). Autoimmune hepatitis is often
responsible for a progressive destruction of the hepatic parenchyma
with a high mortality if left untreated (Johnson et al. (1993)
Hepatology, 18:998). One of the characteristics of autoimmune
hepatitis is the presence of circulating autoantibodies in almost
90% of patients' sera. Such antibodies can be used to identify
subjects who have autoimmune hepatitis.
[0424] Clinical and serological differences between patients have
lead to the classification of AIH into two types. Type 1 is
characterized by the presence of anti-smooth muscle (SMA) and/or
anti-nuclear antibodies (ANA) in patients' sera, while sera from
Type II patients show anti-liver kidney microsomal antibodies type
1 (LKM1) (Homberg et al., (1987) Hepatology, 7:1333; Maggiore et
al. (1993) J. Pediatr. Gastroenterol Nutr. 17:376). A serological
marker, anti-liver cytosol type I antibodies (LC1), has been
identified in 30% of patients with an AIH type II. In addition, LC1
proved to be the only serological marker in 10% of patients tested
(Martini et al. (1988) Hepatology, 8:1662). In one embodiment, the
method of the invention is used to treat AIH.
[0425] 3. Fatty-Liver Disease
[0426] Tumor necrosis factor has been implicated in the
pathophysiology of fatty-liver disease (see Valenti et al., (2002)
Gastroenerology 122:274; Li et al., (2003) Hepatology 37:343).
Fatty-liver disease refers to a disease wherein fat (hepatocytes)
is excessively accumulated in the liver. Fatty liver disease is
believed to be caused by supernutrition, hyperingestion of alcohol,
diabetes and side effects due to administration of pharmaceuticals.
Fatty liver disease can cause severe diseases such as chronic
hepatitis and hepatic cirrhosis. In patients with fatty liver
disease, lipids, particularly neutral fat, accumulate in
hepatocytes to the extent that the amount exceeds the
physiologically permissible range. From a biochemical point of
view, a standard for judgment of fatty liver is that the weight of
neutral fat is about 10% (100 mg/g wet weight) or more of the wet
weight of hepatic tissue. In one embodiment, the method of the
invention is used to treat fatty liver disease.
[0427] 4. Hepatitis B Virus (HBV)
[0428] Tumor necrosis factor has been implicated in the
pathophysiology of hepatitis B virus (see Kasahara et al., (2003)
J. Virol. 77:2469; Wang (2003) World J Gastroenterol. 9:641;
Biermer et al. (2003) J. Virol. 77:4033). The term "hepatitis B
virus" (HBV) is used to describe the virus (serum hepatitis virus)
which produces viral hepatitis type B in humans. This is a viral
disease with a long incubation period (about 50 to 160 days) in
contrast to hepatitis A virus (infectious hepatitis virus) which
has a short incubation period. The hepatitis B virus is usually
transmitted by injection of infected blood or blood derivatives or
merely by use of contaminated needles, lancets or other
instruments. Clinically and pathologically, the disease is similar
to viral hepatitis type A; however, there is no cross-protective
immunity. Viral antigen (HBAg) is found in the serum after
infection.
[0429] Hepatitis B virus infects humans at a very high rate. Most
people who become infected with Hepatitis B get rid of the virus
within 6 months, wherein a short infection is known as an "acute"
case of Hepatitis B. It is estimated that at least about 300
million people are chronic carriers of HBV. Infection with the
virus results in a range of clinical symptoms including minor
flu-like symptoms to death. In one embodiment, the
multiple-variable dose method of the invention is used to treat HBV
infection.
[0430] 5. Hepatotoxicity
[0431] Tumor necrosis factor has been implicated in the
pathophysiology of hepatotoxicity (see Bruccoleri et al. (1997)
Hepatology 25:133; Luster et al. (2000) Ann NY Acad. Sci. 919:214;
Simeonova et al. (2001) Toxicol Appl Pharmacol. 177:112). The term
hepatotoxicity refers to liver damage caused by medications and
other chemicals or drugs. The best indicator for identifying liver
toxicity in a subject is the elevation of certain enzyme
measurements in the blood, such as AST (aspartate
aminotransferase), ALT (alanine aminotransferase), and GOT
(glutamate oxalacetate transaminase).
[0432] Hepatotoxicity can cause permanent injury and death. Initial
symptoms of hepatotoxicity can include acute gastrointestinal
symptoms, e.g., severe diarrhea. The second phase of hepatotoxicity
is characterized by abatement of symptoms. During this apparent
subsidence, biochemical evidence of hepatic injury appears.
Oliguria (decreased urine output) is usual during the second phase.
The third phase, that of overt hepatic damage, becomes clinically
apparent 3 to 5 days after ingestion of the chemical, with the
appearance of jaundice. Renal failure may also occur. The symptoms
of chemically-induced (drug-induced) hepatitis are similar to that
of infectious hepatitis. In one embodiment, the method of the
invention is used to treat hepatotoxicity.
[0433] 6. Liver Failure (e.g. Chronic Liver Failure)
[0434] Tumor necrosis factor has been implicated in the
pathophysiology of liver failure (e.g. chronic liver failure) (see
Takenaka et al., (1998) Dig Dis Sci. 43:887; Nagaki et al. (1999)
J. Hepatol. 31:997; Streetz et al., (2000) Gastroenterology.
119:446. Liver failure, including chronic liver failure, usually
develops over a period of years and is caused by a repeated insult
to the liver (such as alcohol abuse or infection with hepatitis
virus) which slowly damages the organ. Less commonly, liver failure
is acute, and occurs over a period of days or weeks. Causes of
acute liver failure include hepatitis virus infections, drugs,
pregnancy, autoimmune disease, and sudden low blood flow to the
liver. In one embodiment, the method of the invention is used to
treat liver failure.
[0435] 7. Non-Alcoholic Hepatitis, Including NASH
[0436] Tumor necrosis factor has been implicated in the
pathophysiology of non-alcoholic hepatitis, including nonalcoholic
steatohepatitis (see Crespo et al., (2001) Hepatology. 34:1158;
Pessayre et al. (2002) 282(2):G193). The term "nonalcoholic
steatohepatitis" or "NASH" refers to the development of histologic
changes in the liver that are comparable to those induced by
excessive alcohol intake, but in the absence of alcohol abuse. NASH
is characterized by macrovesicular and/or microvesicular steatosis,
lobular and portal inflammation, and occasionally Mallory bodies
with fibrosis and cirrhosis. NASH is also commonly associated with
hyperlipidemia, obesity, and type II diabetes mellitus.
[0437] Additional clinical conditions which characterize hepatic
steatosis and inflammation include excessive fasting, jejunoileal
bypass, total parental nutrition, chronic hepatitis C, Wilson's
disease, and adverse drug effects such as those from
corticosteroids, calcium channel blockers, high dose synthetic
estrogens, methotrexate and amiodarone. Thus, the term
"nonalcoholic steatohepatitis" can be used to describe those
patients who exhibit these biopsy findings, coupled with the
absence of (a) significant alcohol consumption, (b) previous
surgery for weight loss, (c) history of drug use associated with
steatohepatitis, (d) evidence of genetic liver disease or (e)
chronic hepatitis C infection (see, e.g., Ludwig et al., (1980)
Mayo Clin. Proc. 55:434; Powell et al. (1990) Hepatol. 11:74). In
one embodiment, the antibodies obtained using the method of the
invention are used to treat NASH.
N. Skin and Nail Disorders
[0438] Tumor necrosis factor has been implicated in the
pathophysiology of skin and nail disorders. In one embodiment,
antibodies obtained using the method of the invention are
administered to treat skin and nail disorders. The term "skin
disorder" or "skin disease" as used interchangeably herein, refers
to abnormalities, other than injury wounds, of the skin which have
induced a state of inflammation. In one embodiment, the skin
disorder of the invention is an inflammatory skin disorder, wherein
the skin is characterized by capillary dilatation, leukocytic
infiltration, redness, heat, and/or pain. Examples of skin
disorders include, but are not limited to, psoriasis, pemphigus
vulgaris, scleroderma, atopic dermatitis, sarcoidosis, erythema
nodosum, hidradenitis suppurative, lichen planus, Sweet's syndrome,
and vitiligo. As used herein, the term "skin and nail disorder in
which TNF.alpha. activity is detrimental" is intended to include
skin and/or nail disorders and other disorders in which the
presence of TNF.alpha. in a subject suffering from the disorder has
been shown to be or is suspected of being either responsible for
the pathophysiology of the disorder or a factor that contributes to
a worsening of the disorder, e.g., psoriasis. Accordingly, skin and
nail disorders in which TNF.alpha. activity is detrimental are
disorders in which inhibition of TNF.alpha. activity is expected to
alleviate the symptoms and/or progression of the disorder. The use
of the antibodies, antibody portions, and other TNF.alpha.
inhibitors of the invention in the treatment of specific skin and
nail disorders is discussed further below. In certain embodiments,
the treatment method of the invention is performed in combination
with another therapeutic agent, as described below. In one
embodiment, the antibodies obtained using the method of the
invention comprising administering a TNF.alpha. antibody in
combination with another therapeutic agent is used for the
treatment of psoriasis and the treatment of psoriasis associated
with arthritis.
[0439] 1. Psoriasis
[0440] Tumor necrosis factor has been implicated in the
pathophysiology of psoriasis (Takematsu et al. (1989) Arch Dermatol
Res. 281:398; Victor and Gottlieb (2002) J Drugs Dermatol. 1:264).
The term "psoriasis" as used herein, refers to skin disorders
associated with epidermal hyperplasia. Example of psoriasis
include, but are not limited to, chronic plaque psoriasis, guttate
psoriasis, inverse psoriasis, pustular psoriasis, psoriasis
vulgaris, and erythrodermic psoriasis. Psoriasis can also be
associated with other inflammatory disorders, including
inflammatory bowel disease (IBD) and rheumatoid arthritis (RA).
[0441] Psoriasis is described as a skin inflammation (irritation
and redness) characterized by frequent episodes of redness,
itching, and thick, dry, silvery scales on the skin. In particular,
lesions are formed which involve primary and secondary alterations
in epidermal proliferation, inflammatory responses of the skin, and
an expression of regulatory molecules such as lymphokines and
inflammatory factors. Psoriatic skin is morphologically
characterized by an increased turnover of epidermal cells,
thickened epidermis, abnormal keratinization, inflammatory cell
infiltrates into the epidermis and polymorphonuclear leukocyte and
lymphocyte infiltration into the epidermis layer resulting in an
increase in the basal cell cycle. Psoriasis often involves the
nails, which frequently exhibit pitting, separation of the nail,
thickening, and discoloration. Psoriasis is often associated with
other inflammatory disorders, for example arthritis, including
rheumatoid arthritis, inflammatory bowel disease (IBD), and Crohn's
disease. Approximately one third of subjects with psoriasis also
have psoriatic arthritis (PsA) which, as described above, causes
stiffness, swelling of the joints, pain, and reduced range of
motion (Greaves et al. (1995) N. Eng. J. Med. 332:581).
[0442] Evidence of psoriasis is most commonly seen on the trunk,
elbows, knees, scalp, skin folds, or fingernails, but it may affect
any or all parts of the skin. Normally, it takes about a month for
new skin cells to move up from the lower layers to the surface. In
psoriasis, this process takes only a few days, resulting in a
build-up of dead skin cells and formation of thick scales. Symptoms
of psoriasis include: skin patches, that are dry or red, covered
with silvery scales, raised patches of skin, accompanied by red
borders, that may crack and become painful, and that are usually
located on the elbows, knees, trunk, scalp, and hands; skin
lesions, including pustules, cracking of the skin, and skin
redness; joint pain or aching which may be associated with of
arthritis, e.g., psoriatic arthritis.
[0443] Treatment for psoriasis often includes a topical
corticosteroids, vitamin D analogs, and topical or oral retinoids,
or combinations thereof. In one embodiment, the TNF.alpha. antibody
of the invention is administered in combination with or the
presence of one of these common treatments. Additional therapeutic
agents which can be combined with the TNF.alpha. antibody obtained
using the methods of the invention for treatment of psoriasis are
described in more detail below.
[0444] The diagnosis of psoriasis is usually based on the
appearance of the skin. Additionally a skin biopsy, or scraping and
culture of skin patches may be needed to rule out other skin
disorders. An x-ray may be used to check for psoriatic arthritis if
joint pain is present and persistent.
[0445] Improvements in psoriasis in a subject can be monitored by
the subject's Psoriasis Area and Severity Index Score (PASI). The
method for determining the PASI has been described in Fredriksson
and Pettersson (1978) Dermatologica 157:238 and Marks et al. (1989)
Arch Dermatol 125:235. Briefly, the index is based on evaluation of
four anatomic sites, including the head, upper extremities, trunk,
and lower extremities, for erythema, induration, and desquamation
using a 5 point scale (0=no symptoms; 1=slight; 2=moderate;
3=marked; 4=very marked). Based on the extent of lesions in a given
anatomic site, the area affected is assigned a numerical value
(0=0; 1=<10%; 2=10-29%; 3=30-49%; 4=50-69%; 5=70=89%;
6=90-100%). The PASI score is then calculated, wherein the possible
range of PASI score is 0.0 to 72.0 with the highest score
representing complete erythroderma of the severest degree.
[0446] In one embodiment of the invention, a TNF.alpha. antibody is
used for the treatment of psoriasis, including chronic plaque
psoriasis, guttate psoriasis, inverse psoriasis, pustular
psoriasis, pemphigus vulgaris, erythrodermic psoriasis, psoriasis
associated with inflammatory bowel disease (IBD), and psoriasis
associated with rheumatoid arthritis (RA). In another embodiment, a
TNF.alpha. antibody, such as adalimumab, is used to treat subjects
who have psoriasis in combination with PsA. Specific types of
psoriasis included in the treatment methods of the invention are
described in detail below:
[0447] a. Chronic Plaque Psoriasis
[0448] Tumor necrosis factor has been implicated in the
pathophysiology of chronic plaque psoriasis (Asadullah et al.
(1999) Br J Dermatol. 141:94). Chronic plaque psoriasis (also
referred to as psoriasis vulgaris) is the most common form of
psoriasis. Chronic plaque psoriasis is characterized by raised
reddened patches of skin, ranging from coin-sized to much larger.
In chronic plaque psoriasis, the plaques may be single or multiple,
they may vary in size from a few millimeters to several
centimeters. The plaques are usually red with a scaly surface, and
reflect light when gently scratched, creating a "silvery" effect.
Lesions (which are often symmetrical) from chronic plaque psoriasis
occur all over body, but with predilection for extensor surfaces,
including the knees, elbows, lumbosacral regions, scalp, and nails.
Occasionally chronic plaque psoriasis can occur on the penis, vulva
and flexures, but scaling is usually absent. Diagnosis of patients
with chronic plaque psoriasis is usually based on the clinical
features described above. In particular, the distribution, color
and typical silvery scaling of the lesion in chronic plaque
psoriasis are characteristic of chronic plaque psoriasis.
[0449] b. Guttate Psoriasis
[0450] Guttate psoriasis refers to a form of psoriasis with
characteristic water drop shaped scaly plaques. Flares of guttate
psoriasis generally follow an infection, most notably a
streptococcal throat infection. Diagnosis of guttate psoriasis is
usually based on the appearance of the skin, and the fact that
there is often a history of recent sore throat.
[0451] c. Inverse Psoriasis
[0452] Inverse psoriasis is a form of psoriasis in which the
patient has smooth, usually moist areas of skin that are red and
inflammed, which is unlike the scaling associated with plaque
psoriasis. Inverse psoriasis is also referred to as intertiginous
psoriasis or flexural psoriasis. Inverse psoriasis occurs mostly in
the armpits, groin, under the breasts and in other skin folds
around the genitals and buttocks, and, as a result of the locations
of presentation, rubbing and sweating can irritate the affected
areas.
[0453] d. Pustular Psoriasis
[0454] Pustular psoriasis, also referred to as palmar plantar
psoriasis, is a form of psoriasis that causes pus-filled blisters
that vary in size and location, but often occur on the hands and
feet. The blisters may be localized, or spread over large areas of
the body. Pustular psoriasis can be both tender and painful, can
cause fevers.
[0455] e. Other Psoriasis Disorders
[0456] Other examples of psoriatic disorders which can be treated
with the TNF.alpha. antibody obtained using the methods of the
invention include erythrodermic psoriasis, vulgaris, psoriasis
associated with IBD, and psoriasis associated with arthritis,
including rheumatoid arthritis.
[0457] 2. Pemphigus Vulgaris
[0458] Pemphigus vulgaris is a serious autoimmune systemic
dermatologic disease that often affects the oral mucous membrane
and skin. The pathogenesis of pemphigus vulgaris is thought to be
an autoimmune process that is directed at skin and oral mucous
membrane desmosomes. Consequentially, cells do not adhere to each
other. The disorder manifests as large fluid-filled, rupture-prone
bullae, and has a distinctive histologic appearance.
Anti-inflammatory agents are the only effective therapy for this
disease which has a high mortality rate. Complications that arise
in patients suffering from pemphigus vulgaris are intractable pain,
interference with nutrition and fluid loss, and infections.
[0459] 3. Atopic Dermatitis/Eczema
[0460] Atopic dermatitis (also referred to as eczema) is a chronic
skin disorder categorized by scaly and itching plaques. People with
eczema often have a family history of allergic conditions like
asthma, hay fever, or eczema. Atopic dermatitis is a
hypersensitivity reaction (similar to an allergy) which occurs in
the skin, causing chronic inflammation. The inflammation causes the
skin to become itchy and scaly. Chronic irritation and scratching
can cause the skin to thicken and become leathery-textured.
Exposure to environmental irritants can worsen symptoms, as can
dryness of the skin, exposure to water, temperature changes, and
stress.
[0461] Subjects with atopic dermatitis can be identified by certain
symptoms, which often include intense itching, blisters with oozing
and crusting, skin redness or inflammation around the blisters,
rash, dry, leathery skin areas, raw areas of the skin from
scratching, and ear discharges/bleeding.
[0462] 4. Sarcoidosis
[0463] Sarcoidosis is a disease in which granulomatous inflammation
occurs in the lymph nodes, lungs, liver, eyes, skin, and/or other
tissues. Sarcoidosis includes cutaneous sarcoidosis (sarcoidosis of
the skin) and nodular sarcoidosis (sarcoidosis of the lymph nodes).
Patients with sarcoidosis can be identified by the symptoms, which
often include general discomfort, uneasiness, or an ill feeling;
fever; skin lesions.
[0464] 5. Erythema Nodosum
[0465] Erythema nodosum refers to an inflammatory disorder that is
characterized by tender, red nodules under the skin, typically on
the anterior lower legs. Lesions associated with erythema nodosum
often begin as flat, but firm, hot red painful lumps (approximately
an inch across). Within a few days the lesions may become purplish,
and then over several weeks fade to a brownish flat patch.
[0466] In some instances, erythema nodosum may be associated with
infections including, streptococcus, coccidioidomycosis,
tuberculosis, hepatitis B, syphilis, cat scratch disease,
tularemia, yersinia, leptospirosis psittacosis, histoplasmosis,
mononucleosis (EBV). In other instances, erythema nodosum may be
associated with sensitivity to certain medications including,
oralcontraceptives, penicillin, sulfonamides, sulfones,
barbiturates, hydantoin, phenacetin, salicylates, iodides, and
progestin. Erythema nodosum is often associated with other
disorders including, leukemia, sarcoidosis, rheumatic fever, and
ulcerative colitis.
[0467] Symptoms of erythema nodosum usually present themselves on
the shins, but lesions may also occur on other areas of the body,
including the buttocks, calves, ankles, thighs and upper
extremities. Other symptoms in subjects with erythema nodosum can
include fever and malaise.
[0468] 6. Hidradenitis Suppurative
[0469] Hidradenitis suppurativa refers to a skin disorder in which
swollen, painful, inflamed lesions or lumps develop in the groin
and sometimes under the arms and under the breasts. Hidradenitis
suppurativa occurs when apocrine gland outlets become blocked by
perspiration or are unable to drain normally because of incomplete
gland development. Secretions trapped in the glands force
perspiration and bacteria into surrounding tissue, causing
subcutaneous induration, inflammation, and infection. Hidradenitis
suppurativa is confined to areas of the body that contain apocrine
glands. These areas are the axillae, areola of the nipple, groin,
perineum, circumanal, and periumbilical regions.
[0470] 7. Lichen Planus
[0471] Tumor necrosis factor has been implicated in the
pathophysiology of lichen planus (Sklavounou et al. (2000) J Oral
Pathol Med. 29:370). Lichen planus refers to a disorder of the skin
and the mucous membranes resulting in inflammation, itching, and
distinctive skin lesions. Lichen planus may be associated with
hepatitis C or certain medications.
[0472] 8. Sweet's Syndrome
[0473] Inflammatory cytokines, including tumor necrosis factor,
have been implicated in the pathophysiology of Sweet's syndrome
(Reuss-Borst et al. (1993) Br J Haematol. 84:356). Sweet's
syndrome, which was described by R. D. Sweet in 1964, is
characterized by the sudden onset of fever, leukocytosis, and
cutaneous eruption. The eruption consists of tender, erythematous,
well-demarcated papules and plaques which show dense neutrophilic
infiltrates microscopically. The lesions may appear anywhere, but
favor the upper body including the face. The individual lesions are
often described as pseudovesicular or pseudopustular, but may be
frankly pustular, bullous, or ulcerative. Oral and eye involvement
(conjunctivitis or episcleritis) have also been frequently reported
in patients with Sweet's syndrome. Leukemia has also been
associated with Sweet's syndrome.
[0474] 9. Vitiligo
[0475] Vitiligo refers to a skin condition in which there is loss
of pigment from areas of skin resulting in irregular white patches
with normal skin texture. Lesions characteristic of vitiligo appear
as flat depigmented areas. The edges of the lesions are sharply
defined but irregular. Frequently affected areas in subjects with
vitiligo include the face, elbows and knees, hands and feet, and
genitalia.
[0476] 10. Scleroderma
[0477] Tumor necrosis factor has been implicated in the
pathophysiology of scleroderma (Tutuncu et al. (2002) Clin Exp
Rheumatol. 20(6 Suppl 28):S146; Mackiewicz et al. (2003) Clin Exp
Rheumatol. 21:41; Murota et al. (2003) Arthritis Rheum. 48:1117).
Scleroderma refers to a a diffuse connective tissue disease
characterized by changes in the skin, blood vessels, skeletal
muscles, and internal organs. Scleroderma is also referred to as
CREST syndrome or progressive systemic sclerosis, and usually
affects people between the ages 30-50. Women are affected more
often than men.
[0478] The cause of scleroderma is unknown. The disease may produce
local or systemic symptoms. The course and severity of the disease
varies widely in those affected. Excess collagen deposits in the
skin and other organs produce the symptoms. Damage to small blood
vessels within the skin and affected organs also occurs. In the
skin, ulceration, calcification, and changes in pigmentation may
occur. Systemic features may include fibrosis and degeneration of
the heart, lungs, kidneys and gastrointestinal tract.
[0479] Patients suffering from scleroderma exhibit certain clinical
features, including, blanching, blueness, or redness of fingers and
toes in response to heat and cold (Raynaud's phenomenon), pain,
stiffness, and swelling of fingers and joints, skin thickening and
shiny hands and forearm, esophageal reflux or heartburn, difficulty
swallowing, and shortness of breath. Other clinical symptoms used
to diagnose scleroderma include, an elevated erythrocyte
sedimentation rate (ESR), an elevated rheumatoid factor (RF), a
positive antinuclear antibody test, urinalysis that shows protein
and microscopic blood, a chest X-ray that may show fibrosis, and
pulmonary function studies that show restrictive lung disease.
[0480] 11. Nail Disorders
[0481] Nail disorders include any abnormality of the nail. The term
"nail disorder" or "nail disease" as used herein, refers to
conditions wherein the fingernails or toenails to abnormal color,
shape, texture, or thickness. Specific nail disorders include, but
are not limited to, pitting, koilonychia, Beau's lines, spoon
nails, onycholysis, yellow nails, pterygium (seen in lichen
planus), and leukonychia. Pitting is characterised by the presence
of small depressions on the nail surface. Ridges or linear
elevations can develop along the nail occurring in a "lengthwise"
or "crosswise" direction. Beau's lines are linear depressions that
occur "crosswise" (transverse) in the fingernail. Leukonychia
describes white streaks or spots on the nails. Koilonychia is an
abnormal shape of the fingernail where the nail has raised ridges
and is thin and concave Koilonychia is often associated with iron
deficiency.
[0482] Nail disorders which can be treated with the TNF.alpha.
antibody of the invention also include psoriatic nails. Psoriatic
nails include changes in nails which are attributable to psoriasis.
In some instances psoriasis may occur only in the nails and nowhere
else on the body. Psoriatic changes in nails range from mild to
severe, generally reflecting the extent of psoriatic involvement of
the nail plate, nail matrix, i.e., tissue from which the nail
grows, nail bed, i.e., tissue under the nail, and skin at the base
of the nail. Damage to the nail bed by the pustular type of
psoriasis can result in loss of the nail. Nail changes in psoriasis
fall into general categories that may occur singly or all together.
In one category of psoriatic nails, the nail plate is deeply
pitted, probably due to defects in nail growth caused by psoriasis.
In another category, the nail has a yellow to yellow-pink
discoloration, probably due to psoriatic involvement of the nail
bed. A third subtype of psoriatic nails are characterized by white
areas which appear under the nail plate. The white areas are
actually air bubbles marking spots where the nail plate is becoming
detached from the nail bed. There may also be reddened skin around
the nail. A fourth category is evidenced by the nail plate
crumbling in yellowish patches, i.e., onychodystrophy, probably due
to psoriatic involvement in the nail matrix. A fifth category is
characterized by the loss of the nail in its entirety due to
psoriatic involvement of the nail matrix and nail bed.
[0483] Antibodies obtained using the method of the invention can
also be used to treat nail disorders often associated with lichen
planus. Nails in subjects with lichen planus often show thinning
and surface roughness of the nail plate with longitudinal ridges or
pterygium.
[0484] The antibodies obtained using the invention can be used to
treat nail disorders, such as those described herein. Often nail
disorders are associated with skin disorders. In one embodiment,
the invention treatment for nail disorders using a TNF.alpha.
antibody. In another embodiment, the nail disorder is associated
with another disorder, including a skin disorder such as psoriasis.
In another embodiment, the disorder associated with a nail disorder
is arthritis, including psoriatic arthritis.
[0485] 12. Other Skin and Nail Disorders
[0486] Antibodies obtained using the method of the invention can be
used to treat other skin and nail disorders, such as chronic
actinic dermatitis, bullous pemphigoid, and alopecia areata.
Chronic actinic dermatitis (CAD) is also referred to as
photosensitivity dermatitis/actinic reticuloid syndrome (PD/AR).
CAD is a condition in which the skin becomes inflamed, particularly
in areas that have been exposed to sunlight or artificial light.
Commonly, CAD patients have allergies to certain substances that
come into contact with their skin, particularly various flowers,
woods, perfumes, sunscreens and rubber compounds. Bullous
pemphigoid refers to a skin disorder characterized by the formation
of large blisters on the trunk and extremities. Alopecia areata
refers to hair loss characterized by round patches of complete
baldness in the scalp or beard.
[0487] O. Vasculitides
[0488] TNF.alpha. has been implicated in the pathophysiology of a
variety of vasculitides, (see e.g., Deguchi et al. (1989) Lancet.
2:745). In one embodiment, the invention provides a
multiple-variable dose method for inhibiting TNF.alpha. activity in
a subject suffering from a vasculitis in which TNF.alpha. activity
is detrimental.
[0489] The term "vasculitis" or "vasculitides" as used
interchangeably herein, refers to a group of disorders which are
characterized by the inflammation of blood vessels. Blood vessels
of all sizes may be affected, from the largest vessel in the body
(the aorta) to the smallest blood vessels in the skin
(capillaries). The size of blood vessel affected varies according
to the specific type of vasculitis. As used herein, the term "a
vasculitis in which TNF.alpha. activity is detrimental" is intended
to include vasculitis in which the presence of TNF.alpha. in a
subject suffering from the disorder has been shown to be or is
suspected of being either responsible for the pathophysiology of
the disorder or a factor that contributes to a worsening of the
disorder. Such disorders may be evidenced, for example, by an
increase in the concentration of TNF.alpha. in a biological fluid
of a subject suffering from the disorder (e.g., an increase in the
concentration of TNF.alpha. in serum, plasma, synovial fluid, etc.
of the subject), which can be detected, for example, using an
anti-TNF.alpha. antibody as described above.
[0490] There are numerous examples of vasculitides in which
TNF.alpha. activity is detrimental, including Behcet's disease. The
use of the antibodies, or antigen-binding portions thereof, for
treatment of specific vasculitides is discussed further below. In
certain embodiments, the antibody, or antibody portion, obtained
using the invention is administered to the subject in combination
with another therapeutic agent, as described below.
[0491] The antibody, or antibody portion, obtained using the
invention may also be used to treat vasculitis in which TNF.alpha.
activity is detrimental, wherein inhibition of TNF.alpha. activity
is expected to alleviate the symptoms and/or progression of the
vasculitis or to prevent the vasculitis. Subjects suffering from or
at risk of developing vasculitis can be identified through clinical
symptoms and tests. For example, subjects with vasculitides often
develop antibodies to certain proteins in the cytoplasm of
neutrophils, antineutrophil cytoplasmic antibodies (ANCA). Thus, in
some instances, vasculitides may be evidenced by tests (e.g.,
ELISA), which measure ANCA presence.
[0492] Vasculitis and its consequences may be the sole
manifestation of disease or it may be a secondary component of
another primary disease. Vasculitis may be confined to a single
organ or it may simultaneously affect several organs. and depending
on the syndrome, arteries and veins of all sizes can be affected.
Vasculitis can affect any organ in the body.
[0493] In vasculitis, the vessel lumen is usually compromised,
which is associated with ischemia of the tissues supplied by the
involved vessel. The broad range of disorders that may result from
this process is due to the fact that any type, size and location of
vessel (e.g., artery, vein, arteriole, venule, capillary) can be
involved. Vasculitides are generally classified according to the
size of the affected vessels, as described below. It should be
noted that some small and large vessel vasculitides may involve
medium-sized arteries; but large and medium-sized vessel
vasculitides do not involve vessels smaller than arteries. Large
vessel disease includes, but is not limited to, giant cell
arteritis, also known as temporal arteritis or cranial arteritis,
polymyalgia rheumatica, and Takayasu's disease or arteritis, which
is also known as aortic arch syndrome, young female arteritis and
Pulseless disease. Medium vessel disease includes, but is not
limited to, classic polyarteritis nodosa and Kawasaki's disease,
also known as mucocutaneous lymph node syndrome. Non-limiting
examples of small vessel disease are Behcet's Syndrome, Wegner's
granulomatosis, microscopic polyangitis, hypersensitivity
vasculitis, also known as cutaneous vasculitis, small vessel
vasculitis, Henoch-Schonlein purpura, allergic granulamotosis and
vasculitis, also known as Churg Strauss syndrome. Other
vasculitides include, but are not limited to, isolated central
nervous system vasculitis, and thromboangitis obliterans, also
known as Buerger's disease. Classic Polyarteritis nodosa (PAN),
microscopic PAN, and allergic granulomatosis are also often grouped
together and are called the systemic necrotizing vasculitides. A
further description of vasculitis is described below:
[0494] 1. Large Vessel Vasculitis
[0495] In one embodiment, the TNF.alpha. antibody obtained using
the invention may be used to treat subjects who have large vessel
vasculitis. The term "large vessel(s)" as used herein, refers to
the aorta and the largest branches directed toward major body
regions. Large vessels include, for example, the aorta, and its
branches and corresponding veins, e.g., the subclavian artery; the
brachiocephalic artery; the common carotid artery; the innonimate
vein; internal and external jugular veins; the pulmonary arteries
and veins; the venae cavae; the renal arteries and veins; the
femoral arteries and veins; and the carotid arteries. Examples of
large vessel vasculitides are described below.
[0496] a. Giant Cell Arteritis (GCA)
[0497] Tumor necrosis factor has been implicated in the
pathophysiology of giant cell arteritis (Sneller (2002) Cleve.
Clin. J. Med. 69:S1140; Schett et al. (2002) Ann. Rheum. Dis.
61:463). Giant cell arteritis (GCA), refers to a vasculitis
involving inflammation and damage to blood vessels, particularly
the large or medium arteries that branch from the external carotid
artery of the neck. GCA is also referred to as temporal arteritis
or cranial arteritis, and is the most common primary vasculitis in
the elderly. It almost exclusively affects individuals over 50
years of age, however, there are well-documented cases of patients
40 years and younger. GCA usually affects extracranial arteries.
GCA can affect the branches of the carotid arteries, including the
temporal artery. GCA is also a systemic disease which can involve
arteries in multiple locations.
[0498] Histopathologically, GCA is a panarteritis with inflammatory
mononuclear cell infiltrates within the vessel wall with frequent
Langhans type giant cell formation. There is proliferation of the
intima, granulomatous inflammation and fragmentation of the
internal elastic lamina. The pathological findings in organs is the
result of ischemia related to the involved vessels.
[0499] Patients suffering from GCA exhibit certain clinical
symptoms, including fever, headache, anemia and high erythrocyte
sedimentation rate (ESR). Other typical indications of GCA include
jaw or tongue claudication, scalp tenderness, constitutional
symptoms, pale optic disc edema (particularly `chalky white` disc
edema), and vision disturbances. The diagnosis is confirmed by
temporal artery biopsy.
[0500] b. Polymyalgia Rheumatica
[0501] Tumor necrosis factor has been implicated in the
pathophysiology of polymyalgia rheumatica (Straub et al. (2002)
Rheumatology (Oxford) 41:423; Uddhammar et al. (1998) Br. J.
Rheumatol. 37:766). Polymyalgia rheumatica refers to a rheumatic
disorder that is associated with moderate to severe muscle pain and
stiffness in the neck, shoulder, and hip, most noticeable in the
morning. IL-6 and IL-1.beta. expression has also been detected in a
majority of the circulating monocytes in patients with the
polymyalgia rheumatica. Polymyalgia rheumatica may occur
independently, or it may coexist with or precede GCA, which is an
inflammation of blood vessels.
[0502] c. Takayasu's Arteritis
[0503] Tumor necrosis factor has been implicated in the
pathophysiology of Takayasu's arteritis (Kobayashi and Numano
(2002) Intern. Med. 41:44; Fraga and Medina (2002) Curr. Rheumatol.
Rep. 4:30). Takayasu's arteritis refers to a vasculitis
characterized by an inflammation of the aorta and its major
branches. Takayasu's arteritis (also known as Aortic arch syndrome,
young female arteritis and Pulseless disease) affects the thoracic
and abdominal aorta and its main branches or the pulmonary
arteries. Fibrotic thickening of the aortic wall and its branches
(e.g., carotid, inominate, and subclavian arteries) can lead to
reduction of lumen size of vessels that arise from the aortic arch.
This condition also typically affects the renal arteries.
[0504] Takayasu's arteritis primarily affects young women, usually
aged 20-40 years old, particularly of Asian descent, and may be
manifested by malaise, arthralgias and the gradual onset of
extremity claudication. Most patients have asymmetrically reduced
pulses, usually along with a blood pressure differential in the
arms. Coronary and/or renal artery stenosis may occur.
[0505] The clinical features of Takayasu's arteritis may be divided
into the features of the early inflammatory disease and the
features of the later disease. The clinical features of the early
inflammatory stage of Takayasu's disease are: malaise, low grade
fever, weight loss, myalgia, arthralgia, and erythema multiforme.
Later stages of Takayasu's disease are characterized by fibrotic
stenosis of arteries and thrombosis. The main resulting clinical
features are ischaemic phenomena, e.g. weak and asymmetrical
arterial pulses, blood pressure discrepancy between the arms,
visual disturbance, e.g. scotomata and hemianopia, other
neurological features including vertigo and syncope, hemiparesis or
stroke. The clinical features result from ischaemia due to arterial
stenosis and thrombosis.
[0506] 2. Medium Vessel Disease
[0507] In one embodiment, the TNF.alpha. antibody obtained using
the invention may be used to treat subjects who have medium vessel
vasculitis. The term "medium vessel(s)" is used to refer to those
blood vessels which are the main visceral arteries. Examples of
medium vessels include the mesenteric arteries and veins, the iliac
arteries and veins, and the maxillary arteries and veins. Examples
of medium vessel vasculitides are described below.
[0508] a. Polyarteritis Nodosa
[0509] Tumor necrosis factor has been implicated in the
pathophysiology of polyarteritis nodosa (DiGirolamo et al. (1997)
J. Leukoc. Biol. 61:667). Polyarteritis nodosa, or periarteritis
nodosa refers to vasculitis which is a serious blood vessel disease
in which small and medium-sized arteries become swollen and damaged
because they are attacked by rogue immune cells. Polyarteritis
nodosa usually affects adults more frequently than children. It
damages the tissues supplied by the affected arteries because they
don't receive enough oxygen and nourishment without a proper blood
supply.
[0510] Symptoms which are exhibited in patients with polyarteritis
nodosa generally result from damage to affected organs, often the
skin, heart, kidneys, and nervous system. Generalized symptoms of
polyarteritis nodosa include fever, fatigue, weakness, loss of
appetite, and weight loss. Muscle aches (myalgia) and joint
aches(arthralgia) are common. The skin of subjects with
polyarteritis nodosa may also show rashes, swelling, ulcers, and
lumps (nodular lesions).
[0511] Classic PAN (polyarteritis nodosa) is a systemic arteritis
of small to medium muscular arteritis in which involvement of renal
and visceral arteries is common Abdominal vessels have aneurysms or
occlusions in 50% of PAN patients. Classic PAN does not involve the
pulmonary arteries although the bronchial vessels may be involved.
Granulomas, significant eosinophilia and an allergic diathesis are
not part of the syndrome. Although any organ system may be
involved, the most common manifestations include peripheral
neuropathy, mononeuritis multiplex, intestinal ischemia, renal
ischemia, testicular pain and livedo reticularis.
[0512] b. Kawasaki's Disease
[0513] Tumor necrosis factor has been implicated in the
pathophysiology of Kawasaki's disease (Sundel (2002) Curr.
Rheumatol. Rep. 4:474; Gedalia (2002) Curr. Rheumatol. Rep. 4:25).
Although the cause of Kawasaki's disease is unknown, it is
associated with acute inflammation of the coronary arteries,
suggesting that the tissue damage associated with this disease may
be mediated by proinflammatory agents such as TNF.alpha..
Kawasaki's disease refers to a vasculitis that affects the mucus
membranes, lymph nodes, lining of the blood vessels, and the heart.
Kawasaki's disease is also often referred to as mucocutaneous lymph
node syndrome, mucocutaneous lymph node disease, and infantile
polyarteritis. Subjects afflicted with Kawasaki's disease develop
vasculitis often involving the coronary arteries which can lead to
myocarditis and pericarditis. Often as the acute inflammation
diminishes, the coronary arteries may develop aneurysm, thrombosis,
and lead to myocardial infarction.
[0514] Kawasaki's disease is a febrile systemic vasculitis
associated with edema in the palms and the soles of the feet, with
enlargement of cervical lymph nodes, cracked lips and "strawberry
tongue". Although the inflammatory response is found in vessels
throughout the body, the most common site of end-organ damage is
the coronary arteries. Kawasaki's Disease predominantly affects
children under the age of 5. The highest incidence is in Japan but
is becoming increasingly recognized in the West and is now the
leading cause of acquired heart disease in US children. The most
serious complication of Kawasaki disease is coronary arteritis and
aneurysm formation that occurs in a third of untreated
patients.
[0515] 3. Small Vessel Disease
[0516] In one embodiment, a TNF.alpha. antibody is used to treat
subjects who have small vessel vasculitis. The term "small
vessel(s)" is used to refer to arterioles, venules and capillaries.
Arterioles are arteries that contain only 1 or 2 layers of sooth
muscle cells and are terminal to and continuous with the capillary
network. Venules carry blood from the capillary network to veins
and capillaries connect arterioles and venules. Examples of small
vessel vasculitides are described below.
[0517] a. Behcet's Disease
[0518] Tumor necrosis factor has been implicated in the
pathophysiology of Behcet's disease (Sfikakis (2002) Ann. Rheum.
Dis. 61:ii51-3; Dogan and Farah (2002) Oftalmologia. 52:23).
Behcet's disease is a chronic disorder that involves inflammation
of blood vessels throughout the body. Behcet's disease may also
cause various types of skin lesions, arthritis, bowel inflammation,
and meningitis (inflammation of the membranes of the brain and
spinal cord). As a result of Behcet's disease, the subject with the
disorder may have inflammation in tissues and organs throughout the
body, including the gastrointestinal tract, central nervous system,
vascular system, lungs, and kidneys. Behcet's disease is three
times more common in males than females and is more common in the
eastern Mediterranean and Japan.
[0519] Subjects who have Behcet's disease may show clinical
symptoms including recurrent oral ulcers (resembling canker sores),
recurrent genital ulcers, and eye inflammation. Serum levels of
TNF.alpha., IL-8, IL-1, IL-6 INF-.gamma. and IL-12 are elevated in
Behcet's patients, and the production of these factors has been
shown to be elevated in the monocytes of Behcet's patients (see,
e.g., Inflammatory Disease of Blood Vessels (2001) Marcel Dekker,
Inc., eds. G. S. Hoffman and C. M. Weyand, p. 473).
[0520] b. Wegener's Granulomatosis
[0521] Tumor necrosis factor has been implicated in the
pathophysiology of Wegener's granulomatosis (Marquez et al. (2003)
Curr. Rheumatol. Rep. 5:128; Harman and Margo (1998) Surv.
Ophthalmol. 42:458). Wegener's granulomatosis refers to a
vasculitis that causes inflammation of blood vessels in the upper
respiratory tract (nose, sinuses, ears), lungs, and kidneys.
Wegener's granulomatosis is also referred to as midline
granulomatosis. Wegener's granulomatosis includes a granulomatous
inflammation involving the respiratory tract, and necrotizing
vasculitis affecting small to medium-sized vessels. Subjects who
have Wegener's granulomatosis often also have arthritis (joint
inflammation). Glomerulonephritis may also be present in affected
subjects, but virtually any organ may be involved.
[0522] Patients affected with Wegener's granulomatosis typically
show clinical symptoms comprising recurrent sinusitis or epistaxis,
mucosal ulcerations, otitis media, cough, hemoptysis and dyspnea.
The first symptoms of Wegener's granulomatosis frequently include
upper respiratory tract symptoms, joint pains, weakness, and
tiredness.
[0523] c. Churg-Strauss Syndrome
[0524] Tumor necrosis factor has been implicated in the
pathophysiology of Churg-Strauss syndrome (Gross (2002) Curr. Opin.
Rheumatol. 14:11; Churg (2001) Mod. Pathol. 14:1284). Churg-Strauss
syndrome refers to a vasculitis that is systemic and shows early
manifestation signs of asthma and eosinophilia. Churg-Strauss
syndrome is also referred to as allergic granulomatosis and
angiitis, and occurs in the setting of allergic rhinitis, asthma
and eosinophilia. Sinusitis and pulmonary infiltrates also occur in
Churg-Strauss syndrome, primarily affecting the lung and heart.
Peripheral neuropathy, coronary arteritis and gastrointestinal
involvement are common.
[0525] Patients afflicted with Churg-Strauss syndrome can be
diagnosed according to criteria established by the American College
of Rheumatology (ACR). These criteria were intended to distinguish
CSS from other forms of vasculitis. Not all patients meet every
criterion. Some, in fact, may have only 2 or 3 criteria, yet they
are still classified as Churg-Strauss syndrome. The ACR selected 6
disease features (criteria) as being those that best distinguished
Churg-Strauss syndrome from other vasculitides. These criteria
include: 1) asthma; 2) eosinophilia [>10% on differential WBC
count]; 3) mononeuropathy; 4) transient pulmonary infiltrates on
chest X-rays; 5) paranasal sinus abnormalities; and 6) biopsy
comprising a blood vessel with extravascular eosinophils.
P. Other TNF.alpha.-Related Disorders
[0526] In one embodiment, the invention features a
multiple-variable dose method for treating a TNF.alpha.-related
disorder in which TNF.alpha. activity is detrimental, comprising
administering to a subject a TNF.alpha. antibody, such that said
TNF.alpha.-related disorder is treated. Examples of
TNF.alpha.-related disorders in which TNF.alpha. activity is
detrimental, are discussed further below.
[0527] 1. Juvenile Arthritis
[0528] Tumor necrosis factor has been implicated in the
pathophysiology of juvenile arthritis, including juvenile
rheumatoid arthritis (Grom et al. (1996) Arthritis Rheum. 39:1703;
Mangge et al. (1995) Arthritis Rheum. 8:211). In one embodiment,
the TNF.alpha. antibody of the invention is used to treat juvenile
rheumatoid arthritis.
[0529] The term "juvenile rheumatoid arthritis" or "JRA" as used
herein refers to a chronic, inflammatory disease which occurs
before age 16 that may cause joint or connective tissue damage. JRA
is also referred to as juvenile chronic polyarthritis and Still's
disease.
[0530] JRA causes joint inflammation and stiffness for more than 6
weeks in a child of 16 years of age or less. Inflammation causes
redness, swelling, warmth, and soreness in the joints. Any joint
can be affected and inflammation may limit the mobility of affected
joints. One type of JRA can also affect the internal organs.
[0531] JRA is often classified into three types by the number of
joints involved, the symptoms, and the presence or absence of
certain antibodies found by a blood test. These classifications
help the physician determine how the disease will progress and
whether the internal organs or skin is affected. The
classifications of JRA include the following
[0532] a. Pauciarticular JRA, wherein the patient has four or fewer
joints are affected. Pauciarticular is the most common form of JRA,
and typically affects large joints, such as the knees.
[0533] b. Polyarticular HRA, wherein five or more joints are
affected. The small joints, such as those in the hands and feet,
are most commonly involved, but the disease may also affect large
joints.
[0534] c. Systemic JRA is characterized by joint swelling, fever, a
light skin rash, and may also affect internal organs such as the
heart, liver, spleen, and lymph nodes. Systemic JRA is also
referred to as it Still's disease. A small percentage of these
children develop arthritis in many joints and can have severe
arthritis that continues into adulthood.
[0535] 2. Endometriosis
[0536] Tumor necrosis factor has been implicated in the
pathophysiology of endometriosis, as women with endometriosis have
elevated peritoneal levels of TNF (Eisermann et al. (1988) Fertil
Steril 50:573; Halme (1989) Am J Obstet Gynecol 161:1718; Mori et
al. (1991) Am J Reprod Immunol 26:62; Taketani et al. (1992) Am J
Obstet Gynecol 167:265; Overton et al. (1996) Hum Reprod 1996;
11:380). In one embodiment, the TNF.alpha. antibody may be used to
treat endometriosis. The term "endometriosis" as used herein refers
to a condition in which the tissue that normally lines the uterus
(endometrium) grows in other areas of the body, causing pain,
irregular bleeding, and frequently infertility.
[0537] 3. Prostatitis
[0538] Tumor necrosis factor has been implicated in the
pathophysiology of prostatitis, as men with chronic prostatitis and
chronic pelvic pain have significantly higher levels of TNF and
IL-1 in semen compared to controls (Alexander et al. (1998) Urology
52:744; Nadler et al. (2000) J Urol 164:214; Orhan et al. (2001)
Int J Urol 8:495) Furthermore, in a rat model of prostatitis TNF
levels were also increased in comparison to controls (Asakawa et
al. (2001) Hinyokika Kiyo 47:459; Harris et al. (2000) Prostate
44:25). In one embodiment, the TNF.alpha. antibody of the invention
is used to treat prostatitis.
[0539] The term "prostatitis" as used herein refers to an
inflammation of the prostate. Prostatitis is also referred to as
pelvic pain syndrome. Prostatitis manifests itself in a variety of
forms, including nonbacterial prostatitis, acute prostatitis,
bacterial prostatitis, and acute prostatitis. Acute prostatitis
refers to an inflammation of the prostate gland that develops
suddenly. Acute prostatitis is usually caused by a bacterial
infection of the prostate gland. Chronic prostatitis is an
inflammation of the prostate gland that develops gradually,
continues for a prolonged period, and typically has subtle
symptoms. Chronic prostatitis is also usually caused by a bacterial
infection
[0540] 4. Choroidal Neovascularization
[0541] Tumor necrosis factor has been implicated in the
pathophysiology of choroidal neovascularization. For example, in
surgically excised choroidal neovascular membranes, neovascular
vessels stained positive for both TNF and IL-1 (Oh H et al. (1999)
Invest Ophthalmol V is Sci 40:1891). In one embodiment, the
TNF.alpha. antibody is used to treat choroidal neovascularization.
The term "choroidal neovascularization" as used herein refers to
the growth of new blood vessels that originate from the choroid
through a break in the Bruch membrane into the sub-retinal pigment
epithelium (sub-RPE) or subretinal space. Choroidal
neovascularization (CNV) is a major cause of visual loss in
patients with the condition.
[0542] 5. Sciatica
[0543] Tumor necrosis factor has been implicated in the
pathophysiology of sciatica (Ozaktay et al. (2002) Eur Spine J.
11:467; Brisby et al. (2002) Eur Spine J. 11:62). In one
embodiment, the TNF.alpha. antibody of the invention is used to
treat sciatica. The term "sciatica" as used herein refers to a
condition involving impaired movement and/or sensation in the leg,
caused by damage to the sciatic nerve. Sciatica is also commonly
referred to as neuropathy of the sciatic nerve and sciatic nerve
dysfunction. Sciatica is a form of peripheral neuropathy. It occurs
when there is damage to the sciatic nerve, located in the back of
the leg. The sciatic nerve controls the muscles of the back of the
knee and lower leg and provides sensation to the back of the thigh,
part of the lower leg and the sole of the foot. Sciatica can be
indicative of another disorder, including a lumbar herniated disc,
spinal stenosis, degenerative disc disease, isthmic
spondyloisthesis and piniformis syndrome.
[0544] 6. Sjogren's Syndrome
[0545] Tumor necrosis factor has been implicated in the
pathophysiology of Sjogren's syndrome (Koski et al. (2001) Clin Exp
Rheumatol. 19:131). In one embodiment, the TNF.alpha. antibody of
the invention is used to treat Sjogren's syndrome. The term
"Sjogren's syndrome" as used herein refers to a systemic
inflammatory disorder characterized by dry mouth, decreased
tearing, and other dry mucous membranes, and is often associated
with autoimmune rheumatic disorders, such as rheumatoid arthritis.
Dryness of the eyes and mouth are the most common symptoms of this
syndrome. The symptoms may occur alone, or with symptoms associated
with rheumatoid arthritis or other connective tissue diseases.
There may be an associated enlargement of the salivary glands.
Other organs may become affected. The syndrome may be associated
with rheumatoid arthritis, systemic lupus erythematosus,
scleroderma, polymyositis, and other diseases.
[0546] 7. Uveitis
[0547] Tumor necrosis factor has been implicated in the
pathophysiology of uveitis (Wakefield and Lloyd (1992) Cytokine
4:1; Woon et al. (1998) Curr Eye Res. 17:955). In one embodiment,
the TNF.alpha. antibody of the invention is used to treat uveitis.
The term "uveitis" as used herein refers to an inflammation of the
uvea, which is the layer between the sclera and the retina, which
includes the iris, ciliary body, and the choroid. Uveitis is also
commonly referred to as iritis, pars planitis, chroiditis,
chorioretinitis, anterior uveitis, and posterior uveitis. The most
common form of uveitis is anterior uveitis, which involves
inflammation in the front part of the eye, which is usually
isolated to the iris. This condition is often called iritis. In one
embodiment, the term uveitis refers to an inflammation of the uvea
which excludes inflammation associated with an autoimmune disease,
i.e., excludes autoimmune uveitis.
[0548] 8. Wet Macular Degeneration
[0549] Tumor necrosis factor has been implicated in the
pathophysiology of wet macular degeneration. In one embodiment, the
TNF.alpha. antibody of the invention is used to treat wet macular
degeneration. The term "wet macular degeneration" as used herein
refers to a disorder that affects the macula (the central part of
the retina of the eye) and causes decreased visual acuity and
possible loss of central vision. Patients with wet macular
degeneration develop new blood vessels under the retina, which
causes hemorrhage, swelling, and scar tissue.
[0550] 9. Osteoporosis
[0551] Tumor necrosis factor has been implicated in the
pathophysiology of osteoporosis, (Tsutsumimoto et al. (1999) J Bone
Miner Res. 14:1751). Osteoporosis is used to refer to a disorder
characterized by the progressive loss of bone density and thinning
of bone tissue. Osteoporosis occurs when the body fails to form
enough new bone, or when too much old bone is reabsorbed by the
body, or both. The TNF.alpha. antibody, or antigen-binding fragment
thereof, of the invention can be used to treat osteoporosis.
[0552] 10. Osteoarthritis
[0553] Tumor necrosis factor has been implicated in the
pathophysiology of osteoarthritis, (Venn et al. (1993) Arthritis
Rheum. 36:819; Westacott et al. (1994) J. Rheumatol. 21:1710).
Osteoarthritis (OA) is also referred to as hypertrophic
osteoarthritis, osteoarthrosis, and degenerative joint disease. OA
is a chronic degenerative disease of skeletal joints, which affects
specific joints, commonly knees, hips, hand joints and spine, in
adults of all ages. OA is characterized by a number of the
following manifestations including degeneration and thinning of the
articular cartilage with associated development of "ulcers" or
craters, osteophyte formation, hypertrophy of bone at the margins,
and changes in the synovial membrane and enlargement of affected
joints. Furthermore, osteoarthritis is accompanied by pain and
stiffness, particularly after prolonged activity. The antibody, or
antigen-binding fragment thereof, of the invention can be used to
treat osteoarthritis. Characteristic radiographic features of
osteoarthritis include joint space narrowing, subchondral
sclerosis, osteophytosis, subchondral cyst formation, loose osseous
body (or "joint mouse").
[0554] Medications used to treat osteoarthritis include a variety
of nonsteroidal, anti-inflammatory drugs (NSAIDs). In addition, COX
2 inhibitors, including Celebrex, Vioxx, and Bextra, and
Etoricoxib, are also used to treat OA. Steroids, which are injected
directly into the joint, may also be used to reduce inflammation
and pain. In one embodiment of the invention, TNF.alpha. antibodies
of the invention are administered in combination with a NSAIDs, a
COX2 inhibitor, and/or steroids.
[0555] 11. Other
[0556] The methods of the invention, also can be used to treat
various other disorders in which TNF.alpha. activity is
detrimental. Examples of other diseases and disorders in which
TNF.alpha. activity has been implicated in the pathophysiology, and
thus which can be treated using an antibody, or antibody portion,
of the invention, include inflammatory bone disorders, bone
resorption disease, coagulation disturbances, burns, reperfusion
injury, keloid formation, scar tissue formation, pyrexia,
periodontal disease, obesity, radiation toxicity, age-related
cachexia, Alzheimer's disease, brain edema, inflammatory brain
injury, cancer, chronic fatigue syndrome, dermatomyositis, drug
reactions, such as Stevens-Johnson syndrome and Jarisch-Herxheimer
reaction, edema in and/or around the spinal cord, familial periodic
fevers, Felty's syndrome, fibrosis, glomerulonephritides (e.g.
post-streptococcal glomerulonephritis or IgA nephropathy),
loosening of prostheses, microscopic polyangiitis, mixed connective
tissue disorder, multiple myeloma, cancer and cachexia, multiple
organ disorder, myelo dysplastic syndrome, orchitism osteolysis,
pancreatitis, including acute, chronic, and pancreatic abscess,
polymyositis, progressive renal failure, pseudogout, pyoderma
gangrenosum, relapsing polychondritis, rheumatic heart disease,
sarcoidosis, sclerosing cholangitis, stroke, thoracoabdominal
aortic aneurysm repair (TAAA), TNF receptor associated periodic
syndrome (TRAPS), symptoms related to Yellow Fever vaccination,
inflammatory diseases associated with the ear, chronic ear
inflammation, chronic otitis media with or without cholesteatoma,
pediatric ear inflammation, myotosis, ovarian cancer, colorectal
cancer, therapy associated with induced inflammatory syndrome
(e.g., syndromes following IL-2 administration), and a disorder
associated with a reperfussion injury.
[0557] It is understood that all of the above-mentioned
TNF.alpha.-related disorders include both the adult and juvenile
forms of the disease where appropriate. It is also understood that
all of the above-mentioned disorders include both chronic and acute
forms of the disease. In addition, the multiple-variable dose
methods of the invention can be used to treat each of the
above-mentioned TNF.alpha.-related disorders alone or in
combination with one another, e.g., a subject who is suffering from
uveitis and lupus.
Additional Therapeutic Agents
[0558] The invention pertains to pharmaceutical compositions and
methods of use thereof for the treatment of a TNF.alpha.-related
disorder using a multiple-variable dose regimen. The pharmaceutical
compositions comprise a first agent that prevents or inhibits a
TNF.alpha.-related disorder. The pharmaceutical composition and
methods of use may comprise a second agent that is an active
pharmaceutical ingredient; that is, the second agent is therapeutic
and its function is beyond that of an inactive ingredient, such as
a pharmaceutical carrier, preservative, diluent, or buffer. The
second agent may be useful in treating or preventing
TNF.alpha.-related disorders. The second agent may diminish or
treat at least one symptom(s) associated with the targeted disease.
The first and second agents may exert their biological effects by
similar or unrelated mechanisms of action; or either one or both of
the first and second agents may exert their biological effects by a
multiplicity of mechanisms of action. A pharmaceutical composition
may also comprise a third compound, or even more yet, wherein the
third (and fourth, etc.) compound has the same characteristics of a
second agent.
[0559] It should be understood that the pharmaceutical compositions
described herein may have the first and second, third, or
additional agents in the same pharmaceutically acceptable carrier
or in a different pharmaceutically acceptable carrier for each
described embodiment. It further should be understood that the
first, second, third and additional agent may be administered
simultaneously or sequentially within described embodiments.
Alternatively, a first and second agent may be administered
simultaneously, and a third or additional agent may be administered
before or after the first two agents.
[0560] The combination of agents used within the methods and
pharmaceutical compositions described herein may have a therapeutic
additive or synergistic effect on the condition(s) or disease(s)
targeted for treatment. The combination of agents used within the
methods or pharmaceutical compositions described herein also may
reduce a detrimental effect associated with at least one of the
agents when administered alone or without the other agent(s) of the
particular pharmaceutical composition. For example, the toxicity of
side effects of one agent may be attenuated by another agent of the
composition, thus allowing a higher dosage, improving patient
compliance, and improving therapeutic outcome. The additive or
synergistic effects, benefits, and advantages of the compositions
apply to classes of therapeutic agents, either structural or
functional classes, or to individual compounds themselves.
[0561] Supplementary active compounds can also be incorporated into
the compositions. In certain embodiments, an antibody or antibody
portion of the invention is coformulated with and/or coadministered
with one or more additional therapeutic agents that are useful for
treating TNF.alpha.-related disorder in which TNF.alpha. activity
is detrimental. For example, an anti-hTNF.alpha. antibody, antibody
portion, or other TNF.alpha. inhibitor of the invention may be
coformulated and/or coadministered with one or more additional
antibodies that bind other targets (e.g., antibodies that bind
other cytokines or that bind cell surface molecules), one or more
cytokines, soluble TNF.alpha. receptor (see e.g., PCT Publication
No. WO 94/06476) and/or one or more chemical agents that inhibit
hTNF.alpha. production or activity (such as cyclohexane-ylidene
derivatives as described in PCT Publication No. WO 93/19751).
Furthermore, one or more antibodies or other TNF.alpha. inhibitors
of the invention may be used in combination with two or more of the
foregoing therapeutic agents. Such combination therapies may
advantageously utilize lower dosages of the administered
therapeutic agents, thus avoiding possible toxicities or
complications associated with the various monotherapies. Specific
therapeutic agent(s) are generally selected based on the particular
TNF.alpha.-related disorder being treated, as discussed below.
[0562] Nonlimiting examples of therapeutic agents with which an
antibody, antibody portion, or other TNF.alpha. inhibitor can be
combined in a multiple variable dose method of treatment of the
invention include the following: non-steroidal anti-inflammatory
drug(s) (NSAIDs); cytokine suppressive anti-inflammatory drug(s)
(CSAIDs); CDP-571/BAY-10-3356 (humanized anti-TNF.alpha. antibody;
Celltech/Bayer); cA2/infliximab (chimeric anti-TNF.alpha. antibody;
Centocor); 75 kdTNFR-IgG/etanercept (75 kD TNF receptor-IgG fusion
protein; Immunex; see e.g., Arthritis & Rheumatism (1994) Vol.
37, 5295; J. Invest. Med. (1996) Vol. 44, 235A); 55 kdTNF-IgG (55
kD TNF receptor-IgG fusion protein; Hoffmann-LaRoche);
IDEC-CE9.1/SB 210396 (non-depleting primatized anti-CD4 antibody;
IDEC/SmithKline; see e.g., Arthritis & Rheumatism (1995) Vol.
38, S185); DAB 486-IL-2 and/or DAB 389-IL-2 (IL-2 fusion proteins;
Seragen; see e.g., Arthritis & Rheumatism (1993) Vol. 36,
1223); Anti-Tac (humanized anti-IL-2R.alpha.; Protein Design
Labs/Roche); IL-4 (anti-inflammatory cytokine; DNAX/Schering);
IL-10 (SCH 52000; recombinant IL-10, anti-inflammatory cytokine;
DNAX/Schering); IL-4; IL-10 and/or IL-4 agonists (e.g., agonist
antibodies); IL-1RA (IL-1 receptor antagonist; Synergen/Amgen);
anakinra (Kineret.RTM./Amgen); TNF-bp/s-TNF (soluble TNF binding
protein; see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9
(supplement), 5284; Amer. J. Physiol.--Heart and Circulatory
Physiology (1995) Vol. 268, pp. 37-42); R973401 (phosphodiesterase
Type IV inhibitor; see e.g., Arthritis & Rheumatism (1996) Vol.
39, No. 9 (supplement), S282); MK-966 (COX-2 Inhibitor; see e.g.,
Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),
S81); Iloprost (see e.g., Arthritis & Rheumatism (1996) Vol.
39, No. 9 (supplement), S82); methotrexate; thalidomide (see e.g.,
Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),
5282) and thalidomide-related drugs (e.g., Celgen); leflunomide
(anti-inflammatory and cytokine inhibitor; see e.g., Arthritis
& Rheumatism (1996) Vol. 39, No. 9 (supplement), 5131;
Inflammation Research (1996) Vol. 45, pp. 103-107); tranexamic acid
(inhibitor of plasminogen activation; see e.g., Arthritis &
Rheumatism (1996) Vol. 39, No. 9 (supplement), S284); T-614
(cytokine inhibitor; see e.g., Arthritis & Rheumatism (1996)
Vol. 39, No. 9 (supplement), S282); prostaglandin E1 (see e.g.,
Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),
S282); Tenidap (non-steroidal anti-inflammatory drug; see e.g.,
Arthritis & Rheumatism (1996) Vol. 39, No. 9 (supplement),
S280); Naproxen (non-steroidal anti-inflammatory drug; see e.g.,
Neuro Report (1996) Vol. 7, pp. 1209-1213); Meloxicam
(non-steroidal anti-inflammatory drug); Ibuprofen (non-steroidal
anti-inflammatory drug); Piroxicam (non-steroidal anti-inflammatory
drug); Diclofenac (non-steroidal anti-inflammatory drug);
Indomethacin (non-steroidal anti-inflammatory drug); Sulfasalazine
(see e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9
(supplement), S281); Azathioprine (see e.g., Arthritis &
Rheumatism (1996) Vol. 39, No. 9 (supplement), S281); ICE inhibitor
(inhibitor of the enzyme interleukin-1.beta. converting enzyme);
zap-70 and/or lck inhibitor (inhibitor of the tyrosine kinase
zap-70 or lck); VEGF inhibitor and/or VEGF-R inhibitor (inhibitos
of vascular endothelial cell growth factor or vascular endothelial
cell growth factor receptor; inhibitors of angiogenesis);
corticosteroid anti-inflammatory drugs (e.g., SB203580);
TNF-convertase inhibitors; anti-IL-12 antibodies; anti-IL-18
antibodies; interleukin-11 (see e.g., Arthritis & Rheumatism
(1996) Vol. 39, No. 9 (supplement), S296); interleukin-13 (see
e.g., Arthritis & Rheumatism (1996) Vol. 39, No. 9
(supplement), S308); interleukin-17 inhibitors (see e.g., Arthritis
& Rheumatism (1996) Vol. 39, No. 9 (supplement), S 120); gold;
penicillamine; chloroquine; hydroxychloroquine; chlorambucil;
cyclosporine; cyclophosphamide; total lymphoid irradiation;
anti-thymocyte globulin; anti-CD4 antibodies; CD5-toxins;
orally-administered peptides and collagen; lobenzarit disodium;
Cytokine Regulating Agents (CRAB) HP228 and HP466 (Houghten
Pharmaceuticals, Inc.); ICAM-1 antisense phosphorothioate
oligodeoxynucleotides (ISIS 2302; Isis Pharmaceuticals, Inc.);
soluble complement receptor 1 (TP10; T Cell Sciences, Inc.);
prednisone; orgotein; glycosaminoglycan polysulphate; minocycline;
anti-IL2R antibodies; marine and botanical lipids (fish and plant
seed fatty acids; see e.g., DeLuca et al. (1995) Rheum. Dis. Clin.
North Am. 21:759-777); auranofin; phenylbutazone; meclofenamic
acid; flufenamic acid; intravenous immune globulin; zileuton;
azaribine; mycophenolic acid (RS-61443); tacrolimus (FK-506);
sirolimus (rapamycin); amiprilose (therafectin); cladribine
(2-chlorodeoxyadenosine); methotrexate; antivirals; and immune
modulating agents. Any of the above-mentioned agents can be
administered in combination with the TNF.alpha. antibody of the
invention to treat an TNF.alpha.-related disorder using the
multiple variable dose or single dose method of treatments of the
invention.
[0563] In one embodiment, the TNF.alpha. antibody of the invention
is administered in combination with one of the following agents for
the treatment of rheumatoid arthritis using the multiple variable
dose method of treatment of the invention: small molecule inhibitor
of KDR (ABT-123), small molecule inhibitor of Tie-2; methotrexate;
prednisone; celecoxib; folic acid; hydroxychloroquine sulfate;
rofecoxib; etanercept; infliximab; anakinra (Kineret.RTM./Amgen);
leflunomide; naproxen; valdecoxib; sulfasalazine; ibuprofen;
methylprednisolone; meloxicam; methylprednisolone acetate; gold
sodium thiomalate; aspirin; azathioprine; triamcinolone acetonide;
propoxyphene napsylate/apap; folate; nabumetone; diclofenac;
piroxicam; etodolac; diclofenac sodium; oxaprozin; oxycodone hcl;
hydrocodone bitartrate/apap; diclofenac sodium/misoprostol;
fentanyl; anakinra, human recombinant; tramadol hcl; salsalate;
sulindac; cyanocobalamin/fa/pyridoxine; acetaminophen; alendronate
sodium; prednisolone; morphine sulfate; lidocaine hydrochloride;
indomethacin; glucosamine sulfate/chondroitin; cyclosporine;
sulfadiazine; amitriptyline hcl; oxycodone hcl/acetaminophen;
olopatadine hcl; misoprostol; naproxen sodium; omeprazole;
mycophenolate mofetil; cyclophosphamide; rituximab; IL-1 TRAP; MRA;
CTLA4-IG; IL-18 BP; ABT-874; ABT-325 (anti-IL 18); anti-IL 15;
BIRB-796; SCIO-469; VX-702; AMG-548; VX-740; Roflumilast; IC-485;
CDC-801; and mesopram. In another embodiment, the TNF.alpha.
antibody of the invention is administered using a multiple-variable
dose method for the treatment of a TNF.alpha. related disorder in
combination with one of the above mentioned agents for the
treatment of rheumatoid arthritis. In another embodiment, the
above-mentioned additional agents are used in combination with a
TNF.alpha. antibody in the single dose method of treatment of the
invention.
[0564] In one embodiment, the TNF.alpha. antibody of the invention
is administered using the multiple variable dose regimen in
combination with one of the following agents for the treatment of a
TNF.alpha.-related disorder in which TNF.alpha. activity is
detrimental: anti-IL12 antibody (ABT 874); anti-IL18 antibody (ABT
325); small molecule inhibitor of LCK; small molecule inhibitor of
COT; anti-IL1 antibody; small molecule inhibitor of MK2; anti-CD19
antibody; small molecule inhibitor of CXCR3; small molecule
inhibitor of CCR5; small molecule inhibitor of CCR11 anti-E/L
selectin antibody; small molecule inhibitor of P2X7; small molecule
inhibitor of IRAK-4; small molecule agonist of glucocorticoid
receptor; anti-05a receptor antibody; small molecule inhibitor of
C5a receptor; anti-CD32 antibody; and CD32 as a therapeutic
protein.
[0565] In yet another embodiment, a TNF.alpha. antibody obtained
using the invention may be administered in combination with an
antibiotic or antiinfective agent. Antiinfective agents include
those agents known in the art to treat viral, fungal, parasitic or
bacterial infections. The term, "antibiotic," as used herein,
refers to a chemical substance that inhibits the growth of, or
kills, microorganisms. Encompassed by this term are antibiotic
produced by a microorganism, as well as synthetic antibiotics
(e.g., analogs) known in the art. Antibiotics include, but are not
limited to, clarithromycin (Biaxin.RTM.), ciprofloxacin
(Cipro.RTM.), and metronidazole (Flagyl.RTM.).
[0566] In another embodiment, a TNF.alpha. antibody obtained using
the invention may be administered with an additional therapeutic
agent to treat sciatica or pain. Examples of agents which can be
used to reduce or inhibit the symptoms of sciatica or pain include
hydrocodone bitartrate/apap, rofecoxib, cyclobenzaprine hcl,
methylprednisolone, naproxen, ibuprofen, oxycodone
hcl/acetaminophen, celecoxib, valdecoxib, methylprednisolone
acetate, prednisone, codeine phosphate/apap, tramadol
hcl/acetaminophen, metaxalone, meloxicam, methocarbamol, lidocaine
hydrochloride, diclofenac sodium, gabapentin, dexamethasone,
carisoprodol, ketorolac tromethamine, indomethacin, acetaminophen,
diazepam, nabumetone, oxycodone hcl, tizanidine hcl, diclofenac
sodium/misoprostol, propoxyphene napsylate/apap,
asa/oxycod/oxycodone ter, ibuprofen/hydrocodone bit, tramadol hcl,
etodolac, propoxyphene hcl, amitriptyline hcl, carisoprodol/codeine
phos/asa, morphine sulfate, multivitamins, naproxen sodium,
orphenadrine citrate, and temazepam.
[0567] In yet another embodiment, the TNF.alpha.-related disorder
is treated with a TNF.alpha. antibody obtained using the invention
in combination with hemodialysis.
[0568] In another embodiment, a TNF.alpha. antibody obtained using
the invention may be used in combination with a drug used to treat
Crohn's disease or a Crohn's-related disorder in the multiple
variable dose regimen of the invention. Examples of therapeutic
agents which can be used to treat Crohn's include mesalamine,
prednisone, azathioprine, mercaptopurine, infliximab, budesonide,
sulfasalazine, methylprednisolone sod succ, diphenoxylate/atrop
sulf, loperamide hydrochloride, methotrexate, omeprazole, folate,
ciprofloxacin/dextrose-water, hydrocodone bitartrate/apap,
tetracycline hydrochloride, fluocinonide, metronidazole,
thimerosal/boric acid, hyoscyamine sulfate, cholestyramine/sucrose,
ciprofloxacin hydrochloride, meperidine hydrochloride, midazolam
hydrochloride, oxycodone hcl/acetaminophen, promethazine
hydrochloride, sodium phosphate, sulfamethoxazole/trimethoprim,
celecoxib, polycarbophil, propoxyphene napsylate, hydrocortisone,
multivitamins, balsalazide disodium, codeine phosphate/apap,
colesevelam hcl, cyanocobalamin, folic acid, levofloxacin,
natalizumab, methylprednisolone, interferon-gamma, and sargramostim
(GM-CSF). In one embodiment, methotrexate is administered for the
treatment of Crohn's disease at a dose of 2.5 mg to 30 mg per
week.
[0569] In another embodiment, a TNF.alpha. antibody is administered
in combination with an additional therapeutic agent to treat asthma
in the multiple variable dose regimen of the invention. Examples of
agents which can be used to reduce or inhibit the symptoms of
asthma include the following: albuterol; salmeterol/fluticasone;
sodium; fluticasone propionate; budesonide; prednisone; salmeterol
xinafoate; levalbuterol hcl; sulfate/ipratropium; prednisolone
sodium phosphate; triamcinolone acetonide; beclomethasone
dipropionate; ipratropium bromide; Azithromycin; pirbuterol
acetate; prednisolone; theophylline anhydrous; zafirlukast;
methylprednisolone sod succ; clarithromycin; formoterol fumarate;
influenza virus vaccine; methylprednisolone; trihydrate; allergy
injection; cromolyn sodium; cefprozil; fexofenadine hydrochloride;
flunisolide/menthol; levofloxacin; amoxicillin/clavulanate, inhaler
assist device, guaifenesin, dexamethasone sod phosphate;
moxifloxacin hcl; hyclate; guaifenesin/d-methorphan; gatifloxacin;
pephedrine/cod/chlorphenir; cetirizine hydrochloride; mometasone
furoate; salmeterol xinafoate; benzonatate; cephalexin;
pe/hydrocodone/chlorphenir; cetirizine hcl/pseudoephed;
phenylephrine/cod/promethazine; codeine/promethazine; flunisolide;
dexamethasone; guaifenesin/pseudoephedrine;
chlorpheniramine/hydrocodone; nedocromil sodium; terbutaline
sulfate; epinephrine and methylprednisolone, metaproterenol
sulfate.
[0570] In another embodiment, the TNF.alpha..quadrature.antibody of
the invention is administered in combination with an additional
therapeutic agent to treat COPD. Examples of agents which can be
used to reduce or inhibit the symptoms of COPD include, albuterol
sulfate/ipratropium; ipratropium bromide; salmeterol/fluticasone;
albuterol; salmeterol; xinafoate; fluticasone propionate;
prednisone; theophylline anhydrous; levofloxacin;
methylprednisolone sod succ; montelukast sodium; budesonide;
formoterol fumarate; triamcinolone acetonide; guaifenesin;
azithromycin; beclomethasone; dipropionate; levalbuterol hcl;
flunisolide; sodium; trihydrate; gatifloxacin; zafirlukast;
furoate; amoxicillin/clavulanate; flunisolide/menthol;
chlorpheniramine/hydrocodone; metaproterenol sulfate;
methylprednisolone; ephedrine/cod/chlorphenir; pirbuterol acetate;
-ephedrine/loratadine; terbutaline sulfate; tiotropium bromide;
(R,R)-formoterol; TgAAT; Cilomilast and Roflumilast
[0571] In another embodiment, the TNF.alpha. antibody of the
invention is administered in combination with an additional
therapeutic agent to treat IPF. Examples of agents which can be
used to reduce or inhibit the symptoms of IPF include prednisone;
azathioprine; albuterol; colchicines; sulfate; digoxin; gamma
interferon; methylprednisolone sod succ; furosemide; lisinopril;
nitroglycerin; spironolactone; cyclophosphamide; ipratropium
bromide; actinomycin d; alteplase; fluticasone propionate;
levofloxacin; metaproterenol sulfate; morphine sulfate; oxycodone
hcl; potassium chloride; triamcinolone acetonide; tacrolimus
anhydrous; calcium; interferon-alpha; methotrexate; mycophenolate
mofetil.
[0572] In one embodiment of the invention, a TNF.alpha. antibody is
administered in combination with an agent which is commonly used to
treat spondyloarthropathies. Examples of such agents include
nonsteroidal, anti-inflammatory drugs (NSAIDs), COX 2 inhibitors,
including Celebrex.RTM., Vioxx.RTM., and Bextra.RTM., and
etoricoxib. Physiotherapy is also commonly used to treat
spondyloarthropathies, usually in conjunction with non-steroidal
inflammatory drugs.
[0573] In another embodiment, the TNF.alpha. antibody of the
invention may be administered in combination with an additional
therapeutic agent to treat ankylosing spondylitis. Examples of
agents which can be used to reduce or inhibit the symptoms of
ankylosing spondylitis include ibuprofen, diclofenac and
misoprostol, naproxen, meloxicam, indomethacin, diclofenac,
celecoxib, rofecoxib, sulfasalazine, prednisone, methotrexate,
azathioprine, minocyclin, prednisone, etanercept, and
infliximab.
[0574] In another embodiment, the TNF.alpha. antibody of the
invention is administered in combination with an additional
therapeutic agent to treat psoriatic arthritis. Examples of agents
which can be used to reduce or inhibit the symptoms of psoriatic
arthritis include methotrexate; etanercept; rofecoxib; celecoxib;
folic acid; sulfasalazine; naproxen; leflunomide;
methylprednisolone acetate; indomethacin; hydroxychloroquine
sulfate; sulindac; prednisone; betamethasone diprop augmented;
infliximab; methotrexate; folate; triamcinolone acetonide;
diclofenac; dimethylsulfoxide; piroxicam; diclofenac sodium;
ketoprofen; meloxicam; prednisone; methylprednisolone; nabumetone;
tolmetin sodium; calcipotriene; cyclosporine; diclofenac;
sodium/misoprostol; fluocinonide; glucosamine sulfate; gold sodium
thiomalate; hydrocodone; bitartrate/apap; ibuprofen; risedronate
sodium; sulfadiazine; thioguanine; valdecoxib; alefacept; and
efalizumab.
[0575] In one embodiment the TNF.alpha. inhibitor is administered
following an initial procedure for treating coronary heart disease
in the multiple variable dose regimen of the invention. Examples of
such procedures include, but are not limited to coronary artery
bypass grafting (CABG) and Percutaneous transluminal coronary
balloon angioplasty (PTCA) or angioplasty. In one embodiment, the
TNF.alpha. inhibitor is administered in order to prevent stenosis
from re-occurring. In another embodiment of the invention, the
TNF.alpha. inhibitor is administered in order to prevent or treat
restenosis. The invention also provides a method of treatment,
wherein the TNF.alpha. inhibitor is administered prior to, in
conjunction with, or following the insertion of a stent in the
artery of a subject receiving a procedure for treating coronary
heart disease. In one embodiment the stent is administered
following CABG or PTCA.
[0576] A wide variety of stent grafts may be utilized within the
context of the present invention, depending on the site and nature
of treatment desired. Stent grafts may be, for example, bifurcated
or tube grafts, cylindrical or tapered, self-expandable or
balloon-expandable, unibody, or, modular. Moreover, the stent graft
may be adapted to release the drug at only the distal ends, or
along the entire body of the stent graft. The TNF.alpha. inhibitor
of the invention can also be administered on a stent. In one
embodiment, the TNF.alpha. antibody, including, for example,
adalimumab/D2E7/HUMIRA.RTM. is administered by a drug-eluting
stent.
[0577] The TNF.alpha. antibody can be administered in combination
with an additional therapeutic agent to treat restenosis. Examples
of agents which can be used to treat or prevent restenosis include
sirolimus, paclitaxel, everolimus, tacrolimus, ABT-578, and
acetaminophen.
[0578] The TNF.alpha. antibody of the invention can be administered
in combination with an additional therapeutic agent to treat
myocardial infarction. Examples of agents which can be used to
treat or prevent myocardial infarction include aspirin,
nitroglycerin, metoprolol tartrate, enoxaparin sodium, heparin
sodium, clopidogrel bisulfate, carvedilol, atenolol, morphine
sulfate, metoprolol succinate, warfarin sodium, lisinopril,
isosorbide mononitrate, digoxin, furosemide, simvastatin, ramipril,
tenecteplase, enalapril maleate, torsemide, retavase, losartan
potassium, quinapril hcl/mag carb, bumetanide, alteplase,
enalaprilat, amiodarone hydrochloride, tirofiban hcl m-hydrate,
diltiazem hydrochloride, captopril, irbesartan, valsartan,
propranolol hydrochloride, fosinopril sodium, lidocaine
hydrochloride, eptifibatide, cefazolin sodium, atropine sulfate,
aminocaproic acid, spironolactone, interferon, sotalol
hydrochloride, potassium chloride, docusate sodium, dobutamine hcl,
alprazolam, pravastatin sodium, atorvastatin calcium, midazolam
hydrochloride, meperidine hydrochloride, isosorbide dinitrate,
epinephrine, dopamine hydrochloride, bivalirudin, rosuvastatin,
ezetimibe/simvastatin, avasimibe, abciximab, and cariporide.
[0579] The TNF.alpha. antibody of the invention can be administered
in combination with an additional therapeutic agent to treat
angina. Examples of agents which can be used to treat or prevent
angina include: aspirin; nitroglycerin; isosorbide mononitrate;
atenolol; metoprolol succinate; metoprolol tartrate; amlodipine
besylate; digoxin; dilitiazem hydropchloride; isosorbide dinitrate;
clopidogrel bisulfate; nifedipine; atorvastatin calcium; potassium
chloride; simvastatin; verapamil hcl; furosemide; propranolol hcl;
carvedilo; lisinopril; sprionolactone; hydrochlorothiazide;
enalapril maleate; madolol; ramipril; enoxaparin sodium; heparin
sodium; valsartan; sotalol hydrochloride; fenofibrate; ezetimibe;
bumetanide; losartan potassium; lisinopril/hydrochlorothiazide;
felodipine; captopril; and bisoprolol fumarate.
[0580] In one embodiment of the invention, a TNF.alpha. antibody is
administered in combination with an agent which is commonly used to
treat hepatitis C virus. Examples of such agents include
Interferon-alpha-2a, Interferon-alpha-2b, Interferon-alpha con1,
Interfero-aopha-n1, Pegylated interferon-alpha-2a, Pegylated
interferon-alpha-2b, Ribavirin, Peginterferon alfa-2b and
ribavirin, Ursodeoxycholic Acid, Glycyrrhizic Acid, Thymalfasin,
Maxamine, and VX-497.
[0581] The TNF.alpha. antibody may be administered in combination
with topical corticosteroids, vitamin D analogs, and topical or
oral retinoids, or combinations thereof, for the treatment of
psoriasis. In addition, the TNF.alpha. antibody may be administered
in combination with one of the following agents for the treatment
of psoriasis: small molecule inhibitor of KDR (ABT-123), small
molecule inhibitor of Tie-2, calcipotriene, clobetasol propionate,
triamcinolone acetonide, halobetasol propionate, tazarotene,
methotrexate, fluocinonide, betamethasone diprop augmented,
fluocinolone, acetonide, acitretin, tar shampoo, betamethasone
valerate, mometasone furoate, ketoconazole, pramoxine/fluocinolone,
hydrocortisone valerate, flurandrenolide, urea, betamethasone,
clobetasol propionate/emoll, fluticasone propionate, azithromycin,
hydrocortisone, moisturizing formula, folic acid, desonide, coal
tar, diflorasone diacetate, etanercept, folate, lactic acid,
methoxsalen, hc/bismuth subgal/znox/resor, methylprednisolone
acetate, prednisone, sunscreen, salicylic acid, halcinonide,
anthralin, clocortolone pivalate, coal extract, coal tar/salicylic
acid, coal tar/salicylic acid/sulfur, desoximetasone, diazepam,
emollient, pimecrolimus emollient, fluocinonide/emollient, mineral
oil/castor oil/na lact, mineral oil/peanut oil, petroleum/isopropyl
myristate, psoralen, salicylic acid, soap/tribromsalan,
thimerosal/boric acid, celecoxib, infliximab, alefacept,
efalizumab, tacrolimus, pimecrolimus, PUVA, UVB and other
phototherapy, and sulfasalazine.
[0582] An antibody, antibody portion, may be used in combination
with other agents to treat skin conditions. For example, an
antibody, antibody portion, or other TNF.alpha. inhibitor of the
invention is combined with PUVA therapy. PUVA is a combination of
psoralen (P) and long-wave ultraviolet radiation (UVA) that is used
to treat many different skin conditions. The antibodies, antibody
portions, or other TNF.alpha. inhibitors of the invention can also
be combined with pimecrolimus. In another embodiment, the
antibodies of the invention are used to treat psoriasis, wherein
the antibodies are administered in combination with tacrolimus. In
a further embodiment, tacrolimus and TNF.alpha. inhibitors are
administered in combination with methotrexate and/or cyclosporine.
In still another embodiment, the TNF.alpha. inhibitor of the
invention is administered with excimer laser treatment for treating
psoriasis.
[0583] Nonlimiting examples of other therapeutic agents with which
a TNF.alpha. antibody can be combined to treat a skin or nail
disorder include UVA and UVB phototherapy. Other nonlimiting
examples which can be used in combination with a TNF.alpha.
inhibitor include anti-IL-12 and anti-IL-18 therapeutic agents,
including antibodies.
[0584] In one embodiment, the TNF.alpha. antibody may be
administered in combination with an additional therapeutic agent in
the treatment of Behcet's disease. Additional therapeutic agents
which can be used to treat Behcet's disease include, but are not
limited to, prednisone, cyclophosphamide (Cytoxan), Azathioprine
(also called imuran, methotrexate, timethoprim/sulfamethoxazole
(also called bactrim or septra) and folic acid.
[0585] Any one of the above-mentioned therapeutic agents, alone or
in combination therewith, can be administered to a subject
suffering from a TNF.alpha.-related disorder in which TNF.alpha. is
detrimental, in combination with the TNF.alpha. antibody using a
multiple variable dose treatment regimen of the invention. In one
embodiment, any one of the above-mentioned therapeutic agents,
alone or in combination therewith, can be administered to a subject
suffering from rheumatoid arthritis in addition to a TNF.alpha.
antibody to treat a TNF.alpha.-related disorder. It should be
understood that the additional therapeutic agents can be used in
combination therapy as described above, but also may be used in
other indications described herein wherein a beneficial effect is
desired.
[0586] This invention is further illustrated by the following
examples which should not be construed as limiting. The contents of
all references, patents and published patent applications cited
throughout this application are incorporated herein by
reference
EXAMPLES
Example 1
Purification Procedure for Adalimumab
[0587] In this example, a purification process for purifying a
mixture of adalimumab and host cell proteins (HCPs) was devised,
which process is referred to as process A. In process A, the
adalimumab-HCP mixture was not subjected to a protein A
chromatography step. The first column used in process A was a
cation exchange resin, Fractogel S, to which adalimumab bound while
HCP flowed through. Adalimumab was then eluated from the Fractogel
S column in a first eluate. Next, the first eluate was subjected to
pH viral inactivation to obtain a virally inactivated preparation.
Next, the virally inactivated preparation was applied to an anion
ion exchange resin, a Q sepharose column, to which adalimumab does
not bind, to thereby obtain a first flow through. The first flow
was then applied to a hydrophobic interaction column, a phenyl
sepharose column, to which adalimumab binds and HCP flows through,
to thereby obtain a second eluate. Further processing and packaging
of the second eluate was performed to obtain the final bottled
product.
[0588] In more detail, process A comprises the following steps:
[0589] Step 1:
[0590] Fractogel S column, 100.times.20 cm (157 L), v=175 cm/hr,
Load .ltoreq.30 g protein/L resin per cycle, equilibrated with 20
mM sodium phosphate, 25 mM sodium chloride. After loading of
adalimumab, the column was washed once with equilibration buffer
and eluted with an elution buffer comprising 20 mM sodium
phosphate, 150 mM sodium chloride to obtain the first eluate;
[0591] Step 2:
[0592] Delipid filtration;
[0593] Step 3:
[0594] Ultrafiltration;
[0595] Step 4:
[0596] pH inactivation at pH 3.5 for 1 hour; after inactivation was
complete, pH was adjusted to 6.8 to 7.5, the filter train was
washed with two volumes of 50 mM trolamine; Step 5:
[0597] Q Sepharose FF column, 60.times.30 cm (85 L), v=150 cm/hr,
Load .ltoreq.40 g protein/L resin per cycle, equilibrated an
equilibration buffer comprising 25 mM trolamine, 40 mM sodium
chloride, pH 7.6; flow through obtained;
[0598] Step 6:
[0599] Phenyl Sepharose HP column, 80.times.15 cm (75 L), v=75
cm/hr (elute 37.5 cm/hr), Load 20-40 g protein/L resin per cycle,
equilibrated with an equilibration buffer comprising 20 mM sodium
phosphate, 1.1 M (NH.sub.4).sub.2SO.sub.4, pH 7, washed once with
equilibration buffer and eluted by performing a salt step-gradient
to 11 mM sodium phosphate, 0.625 M (NH.sub.4).sub.2SO.sub.4, pH
7.0, to thereby obtain a second eluate, with fractionation of
product peak if load .ltoreq.35 g protein/L resin;
[0600] Step 7:
[0601] Viral filtration;
[0602] Step 8:
[0603] Final Ultrafiltration/Diafiltration;
[0604] Step 9:
[0605] Final Bottling.
Further details of process A are also described in Example 2
below.
Example 2
Purification of Adalimumab to Improve Yield and Decrease
Impurities
[0606] Modifications were introduced to the capture and fine
purification operations in the manufacturing process of the
antibody adalimumab, namely process A described in Example 1 above.
The modified process is referred to herein as "process B", and
includes the following overall steps: The starting material was the
mixture obtained from the fermentation process using the Chinese
Hamster Ovary (CHO) cell expression system. The mixture was first
separated using cation exchange chromatography, i.e., a Fractogel S
column, where adalimumab was captured on the column (referred to as
"capture"). The load on the Fractogel S column was increased due to
displacement. An improved method of washing the Fractogel S column
was used to decrease the amount of host cell protein (HCP). The
Fractogel S column with bound adalimumab was washed with a
plurality of washes, including an intermediate wash which was a
higher conductivity wash comprising 45% elution buffer and 55%
water for injection (WFI). Following capture and washing,
adalimumab was eluted from the Fractogel S column and the eluate
subjected to anion exchange chromatography, i.e., a Q Sepharose
column. Prior to running the first adalimumab eluate over the anion
exchange column, the eluate was virally inactivated using an
improved method based on pH and conductivity. The adalimumab
preparation was collected in the flow-through of the anion column,
and was subsequently separated further according to hydrophobic
interaction chromatography, i.e., phenyl sepharose column. The
eluate from the phenyl sepharose column was the further processed
for viral filtration, final ultrafiltration, and final bottling
according to standard methods in the art.
[0607] Process B is an improved purification method for achieving
an antibody preparation having a reduced level of HCP and
procathepsin L. The processes described herein were performed using
a 6000 L volume, however, it should be noted that the modifications
described in process B may be used with any volume. A comparison
between the modifications of process A versus process B is provided
in Table 5 (modifications in process B are highlighted in
bold):
TABLE-US-00005 TABLE 5 Comparison of process A and process B Unit
operation Process A Process B Fractogel S column 100 .times. 20 cm
(157 L) 100 .times. 20 cm (157 L) v = 175 cm/hr v = 175 cm/hr Load
.ltoreq.30 g protein/L per cycle Load .ltoreq.35 g protein/L per
cycle Wash 1 = equilibration buffer Wash 1 = equilibration buffer
Elution = elution buffer Wash 2 = 45% elution buffer: 55% WFI
Elution = elution buffer Delipid filtration There are no changes to
this processing step. Ultrafiltration There are no changes to this
processing step. pH Inactivation After inactivation complete, After
inactivation complete, adjust pH to 6.8-7.5 adjust pH to 7.8-8.2
Wash filter train with 2 volumes Wash filter train with of 50 mM
trolamine approximately 2.5 volumes of WFI to achieve conductivity
in the range of 3.9-5.2 mS/cm Q Sepharose FF column 60 .times. 30
cm (85 L) 60 .times. 30 cm (85 L) v = 150 cm/hr v = 150 cm/hr Load
.ltoreq.40 g protein/L resin per Load .ltoreq.40 g protein/L resin
per cycle cycle Phenyl Sepharose HP column 80 .times. 15 cm (75 L)
80 .times. 15 cm (75 L) v = 75 cm/hr (elute 37.5 cm/hr) v = 75
cm/hr (elute 37.5 cm/hr) Load 20-40 g protein/L resin per Load
20-40 g protein/L resin per cycle with fractionation of cycle with
no fractionation of product peak if load .ltoreq.35 g/L product
peak if load .ltoreq.35 g/L resin resin Viral Filtration There are
no changes to this processing step. Final ultrafiltration/ There
are no changes to this processing step. diafiltration Final
bottling There are no changes to this processing step.
The modifications to the various steps in process B are described
in more detail below:
Cation Chromatography
[0608] The primary recovery and capture operations of process B
comprise depth filtration, Fractogel SO.sub.3.sup.- cation exchange
chromatography (Fractogel S), the latter of which serves to capture
adalimumab from the clarified harvest and reduce process-related
impurities (e.g., CHO host cell and medium impurities). A 100 cm
diameter.times.20 cm long column (bed volume 157 L) was used for
this operation. The column was packed with Fractogel S resin (EM
Industries, Hawthorne, N.Y.) and the asymmetry and Height of an
Equivalent Theoretical Plate (HETP) are measured to determine the
quality of the packing. The column was then sanitized with 1.0 M
NaOH for 1 hour, and stored in 0.1 M NaOH until ready for use.
[0609] Cation exchange chromatography can be affected by protein
loading, ionic strength (controlled by filtered harvest dilution),
pH and linear velocity. Protein loading can affect selectivity,
resolution (purity) and yield. Ionic strength (controlled by load
dilution) and pH of the load sample can affect binding capacity,
selectivity, resolution and yield. Linear velocity may affect mass
transport properties, potentially resulting in decreased binding
and resolution at very high flow rates and axial dispersion at very
low flow rates.
[0610] The maximum load to the Fractogel S column was increased to
35 g protein per liter resin. The cation column was equilibrated
with 20 mM sodium phosphate, 25 mM NaCl, pH 7. Following
equilibration, the column is loaded with .ltoreq.35 g protein/L
resin of diluted depth filtrate. One part depth filtrate was
diluted with approximately 1.3 parts of water to reduce the
conductivity to approximately 6.1 mS/cm. The column was then washed
to baseline with equilibration buffer followed by a wash with 9 mM
sodium phosphate, 68 mM NaCl, pH 7 (equivalent to 45% elution
buffer, 55% WFI). The product was eluted from the column in a
single fraction with 20 mM sodium phosphate, 150 mM NaCl, pH 7
(elution buffer). The product pool is collected from 10% full-scale
deflection of the product peak A.sub.280 on both the leading and
trailing edges. The column was cycled as necessary to process the
crude adalimumab. The Fractogel S eluates from each column cycle
were pooled into the same collection tank. Between each cycle, the
column was regenerated with 25 mM sodium phosphate, 1.0 M NaCl, pH
7.
[0611] Studies performed at laboratory scale demonstrated that
efficient recovery of product and reduction in HCP can be achieved
at higher load ranges than the previously established Acceptable
Operating Range (AOR) of 15 to 30 g protein/L resin. Analysis of
adalimumab breakthrough versus column load indicates that the
calculated 5% breakthrough occurs at 38 g/L resin at pH 7.
Therefore, a revised AOR of .ltoreq.35 g protein/L resin was
established for the Fractogel S chromatography step. In sum, the
load limit was also increased to 35 grams protein per liter of
resin to increase process capacity.
[0612] In another set of experiments with the Fractogel resin, the
effect of pH on adalimumab breakthrough versus column load was
examined. In particular, a product breakthrough curve was used to
determine the resin dynamic binding capacity under defined loading
conditions. Table 6 below summarizes the recovery data for the
Fractogel loading capacity study at the previously described pH 7
conditions. Recovery percentage at 10 g/L was normalized to
100%.
TABLE-US-00006 TABLE 6 Recovery data for Fractogel loading capacity
study at pH 7 Loading capacity AYF16G AYF17G Average (g/L) recovery
(%) recovery (%) recovery (%) 10 100 100 100 15 99 99 99 20 98 98
98 25 99 97 98 30 98 96 97 40 95 95 95 50 93 88 91 Fractogel step
yield .gtoreq.50%, BR-068.
[0613] The results at pH 7 show greater than 90% adalimumab
recovery was observed for loading conditions of less than 50 g
adalimumab per liter of Fractogel resin. The adalimumab
breakthrough was plotted versus loading capacity to generate a
theoretical breakthrough curve. At pH 7, the theoretical 10%
breakthrough was found to be at 54 g adalimumab per liter of
Fractogel resin. Also at pH7, the theoretical 5% breakthrough was
found to be at 38 g adalimumab per liter of Fractogel resin,
confirming the results described above.
[0614] A similar study was carried out as described above except
that the load and the first wash pH conditions were adjusted to pH
5. The cation column was equilibrated with 24 mM citric acid and 51
mM sodium phosphate, dibasic, pH 5. Following equilibration, the
column was loaded with up to 80 g protein/L resin of diluted depth
filtrate. The cell culture harvest was pH adjusted to 5.0 with 3M
acidic acid prior to the depth filtration. One part depth filtrate
was diluted with approximately the same volume of water to reduce
the conductivity to approximately 8 to 10 mS/cm. Again, the
adalimumab breakthrough was plotted versus loading capacity to
generate a theoretical breakthrough curve. Under the studied
conditions, the theoretical 10% breakthrough was found to be at
approximately 74 g adalimumab per liter of Fractogel resin. The
theoretical 5% breakthrough was found to be at approximately 73 g
adalimumab per liter of Fractogel resin. Due to the character of
the cation exchange of the resin, lowering the pH of the
chromatography conditions significantly increased adalimumab
dynamic binding capacity. Comparing the breakthrough curves at pH 5
and pH 7, the binding between adalimumab molecule and the Fractogel
resin was observed to be much stronger at lower pH. It was also
found that with the higher loading dynamic capacity at pH 5, better
HCP clearance was achieved. Table 7 below summarizes the data for
the Fractogel loading capacity study vs. the HCP present in the
eluate at the newly tested pH 5 conditions. The data clearly
indicate that under the tested conditions, HCP displacement by
adalimumab had occurred.
TABLE-US-00007 TABLE 7 HCP Present in Fractogel Eluate at Different
Loading Capacities at pH 5 HCP (ng Loading capacity HCP/mg (g/L)
adalimumab) 15 6102 20 6782 25 5767 30 5167 40 3983 60 3207
[0615] Since the analysis of adalimumab breakthrough versus column
load at pH 5 indicated that the calculated 5% breakthrough occurs
at 73 g/L resin, a revised AOR of .ltoreq.70 g protein/L resin can
be established for the Fractogel S chromatography step at pH 5. In
sum, the load limit, which previously was increased to 35 grams
protein per liter of resin at pH 7 (as described above), can be
further increased to 70 grams protein per liter of resin by
lowering of the pH to 5.
Intermediate Wash
[0616] To further reduce the amount of impurities in the adalimumab
preparation, an intermediate wash step was performed prior to
adalimumab elution from the cation column (see Table 8 below). This
additional wash was adjusted relative to the conductivity of the
elution buffer, and helped to improve clearance of HCPs. The
insertion of an intermediate wash step prior to elution reduced the
amount of HCP eluted with adalimumab by over 60% compared to
process A. Parameters investigated included the blend of elution
buffer with water used in the wash (% elution buffer),
conductivity, pH, wash volume, flow rate and resin age. The optimum
wash consists of a blend of 45% elution buffer (20 mM sodium
phosphate, 150 mM sodium chloride, pH 7) and 55% water. Table 8
presents data comparing the level of HCP in the Fractogel S eluate
with and without the additional wash. Fractogel S eluate samples
were assayed for HCP and compared with HCP levels in the eluate
from a pilot-scale Fractogel S process, which incorporated a higher
load and the wash step. The pilot-scale data indicates that the
addition of the second wash step significantly improves the
clearance of HCP by the Fractogel S step.
TABLE-US-00008 TABLE 8 HCP levels in Fractogel S eluate with and
without pre-elution wash step at laboratory scale Column Load Step
g protein/L yield HCP (ng/mg Sample resin (%) adalimumab) Lot A no
2.sup.nd wash.sup.a 25 96 19410 Lot B no 2.sup.nd wash.sup.a 25 96
22992 Lot C no 2.sup.nd wash.sup.a 25 93 21931 Lot D no 2.sup.nd
wash.sup.a 25 97 20037 6000 L load.sup.b, pilot-scale.sup.c with
2.sup.nd 30 95 4914 wash step .sup.aFractogel S eluate sample was
taken from the indicated 6000 L lot and analyzed for HCP content
.sup.bLoad consisted of a blend of filtered harvest from various
lots .sup.cPilot-scale column size is 10 (D) .times. 21 (L) cm;
2.sup.nd wash buffer: 45% elution buffer (20 mM sodium phosphate,
150 mM sodium chloride, pH 7.2), 55% water for injection.
[0617] Typical elution profiles for the Fractogel S chromatography
step for each process are provided in FIG. 1. Process B includes
the above-mentioned intermediate additional wash step prior to
elution, thus the leading edge of the elution peak is sharper with
less early-eluting species detected than that of the previous
process. In sum, an intermediate wash step, just prior to the
elution of adalimumab, was introduced to the Fractogel S step to
improve clearance of process-related impurities, such as HCPs.
Viral Inactivation
[0618] The low pH inactivation step of process B provides a margin
of safety by inactivating potential undetected enveloped viruses
that may be present in the delipid filtrate. The viral-inactivated
pool is subsequently pH-neutralized and filtered to remove
particulates and minimize bioburden. The quality of the adalimumab
during low pH virus inactivation may be affected by pH and the
duration of the low pH incubation. Virus inactivation is dependent
on these same parameters, and it may be affected by the protein
concentration, which may reduce inactivation at high
concentrations. The minimum incubation time at low pH was increased
from 15 minutes to 60 minutes. Analysis of manufacturing samples
taken before and after the low pH step confirmed that adalimumab
can be safely held at pH 3.5 for 1 hour without compromising its
ability to protect murine L929 cells against the cytotoxic effects
of tumor necrosis factor (TNF).
[0619] Following inactivation, the pH and conductivity of the
viral-inactivated eluate were adjusted in accordance with the
equilibration buffer of the following column, e.g., Q Sepharose
column The pH was adjusted to 7.8-8.2, with a target pH of 8.0. In
sum, the pH and conductivity of the viral-inactivated pool, which
serves as the Q Sepharose FF load, was adjusted to match to the pH
and conductivity of the Q Sepharose equilibration buffer.
Anion Chromatography
[0620] The anionic column, i.e., Q Sepharose, step serves to reduce
process-related impurities such as HCP, specifically including
procathepsin L, as well as DNA and insulin. A 60 cm
diameter.times.30 cm long column (bed volume 85 L) was used for Q
Sepharose FF chromatography. The column was packed with Q Sepharose
FF resin (Amersham Pharmacia, Piscataway, N.J.) and asymmetry and
HETP were measured to determine the quality of the packing. The
column was then sanitized with 1.0 M NaOH for 1 hour, and stored in
25 mM sodium phosphate, 20% isopropanol until ready for use.
[0621] Equilibration of the resin was accomplished with 25 mM
trolamine, 40 mM NaCl, pH 8 (equilibration buffer). The maximum
protein loading for this step was .ltoreq.40 g protein/L of resin
per cycle. Process-related impurities adsorbed to the resin, and
adalimumab flowed through the column. The diluted, filtered,
virus-inactivated material was typically processed in two cycles of
approximately equal amounts; additional cycles may be required to
process all available material. Loading and elution were performed
at 150 cm/hr, and the column flow-through is collected when the
A.sub.280 rises above 2% full scale. The column was then washed
with equilibration buffer and the wash was collected until the
A.sub.280 returns to 5% full scale. The wash is pooled with the
flow-through and is designated Q Sepharose FTW. Between cycles, the
column was regenerated with 25 mM sodium phosphate, 1.0 M NaCl, pH
7, and then equilibrated with equilibration buffer.
[0622] Anion exchange chromatography operated in flow-through mode
can be affected by protein loading, ionic strength (conductivity,
which may be controlled by dilution of the low pH inactivation
filtrate), pH and linear velocity. Protein loading can affect
selectivity and yield. Ionic strength and pH of the load sample can
affect binding capacity and selectivity. Linear velocity may affect
mass transport properties, potentially resulting in decreased
binding of process related impurities at very high flow rates and
axial dispersion at very low flow rates. New load conductivity and
pH ranges have been established based on laboratory studies
[0623] Laboratory studies indicated that reduction of HCP by the Q
Sepharose FF step could be enhanced by alterations to the loading
conditions. Parameters investigated included the load pH,
conductivity and grams of protein loaded per L of resin. Adjustment
of the load conductivity and pH to match that of the column
equilibration buffer (5 mS/cm, pH 8), and limiting the load
.ltoreq.40 g adalimumab/L resin result in improved clearance of HCP
and procathepsin L. Table 9 presents laboratory-scale data showing
the reduction in HCP under process A (pH 7.7, conductivity 6.65
mS/cm) and the improved process conditions of pH 8 and conductivity
of 5 mS/cm of process B. Limiting the load on the Q Sepharose
column to 40 g/L of resin provides a four-fold improvement in
clearance of HCP and the additional modifications to the pH and
conductivity of the load yield a three-fold further improvement in
HCP reduction.
TABLE-US-00009 TABLE 9 HCP reduction under varying Q Sepharose FF
load conditions Load Flow- amount Load HCP through Fold G protein/
(ng/mg HCP (ng/mg reduction L resin Load conditions adalimumab)
adalimumab) in HCP 80 pH 7.7, 6.65 mS/cm 726 452 1.6 40 pH 7.7,
6.65 mS/cm 726 114 6.4 40 pH 8.1, 5.08 mS/cm 726 37.6 19.3
[0624] The HCP-reduced flowthrough comprising adalimumab obtained
from the ion exchange column was subsequently used in hydrophobic
interaction chromatography.
Hydrophobic Interaction Chromatography
[0625] The objective of the Phenyl Sepharose HP chromatography
column was to further reduce process-related and product-related
impurities such as host cell proteins and aggregates, respectively.
An 80 cm diameter.times.15 cm long column (bed volume 75 L) was
used for this operation. The column was packed with Phenyl
Sepharose HP resin (Amersham Pharmacia, Piscataway, N.J.) and
asymmetry and HETP were measured to determine the quality of the
packing. The column was then sanitized with 1.0 M NaOH for 1 hour,
and stored in 25 mM Na Phosphate, 20% isopropanol until ready for
use.
[0626] Equilibration of the resin was accomplished with 20 mM
sodium phosphate, 1.1 M (NH.sub.4).sub.2SO.sub.4, pH 7.0
(equilibration buffer). The protein loading for this step was 20 to
40 g protein per L of resin, and two or three chromatography cycles
were required to process the entire quantity of available material.
The column operated at a linear velocity of 75 cm/hr. The Q
Sepharose flowthrough was diluted with an equal volume of 40 mM
sodium phosphate, 2.2 M (NH.sub.4).sub.2SO.sub.4, pH 7.0. Following
loading the column was washed with 20 mM sodium phosphate, 1.1 M
(NH.sub.4).sub.2SO.sub.4, pH 7.0. The product was eluted by
performing a salt step-gradient to 11 mM sodium phosphate, 0.625 M
(NH.sub.4).sub.2SO.sub.4, pH 7.0. Product was collected as the
absorbance rises above 50% UV full scale and continued until
absorbance decreases to less than 20% UV full scale as the peak
tails.
[0627] The process modifications to the Fractogel S and Q Sepharose
FF chromatography steps significantly reduced the burden of
process-related impurity reduction placed upon the Phenyl Sepharose
HP step. As a consequence of the changes, the major function of the
Phenyl Sepharose HP step was the removal of adalimumab
aggregates.
[0628] Process A required that at column loads of 35 g protein/L
resin or higher, product was collected as the UV absorbance rises
above 50% full scale deflection and continues until absorbance
decreases to <20% full scale. At column loadings below 35 g
protein/L resin, the first 0.15 column volume of the eluate peak
was excluded from the collected pool to improve HCP clearance at
this step. The incorporation of the modifications at the previous
chromatography steps in process B alleviated the need for the peak
exclusion at loads below 35 g protein/L resin since the incoming
HCP load was significantly reduced. The reduction in the HCP load
allowed expansion of the load range without fractionation. The
effect of this change permits processing of all material from each
fermentation by the recovery process without Phenyl Sepharose HP
peak cutting
[0629] The linear flow rate for Phenyl Sepharose operation was
investigated at laboratory scale. The adalimumab load was held
constant and flow rates of 25 to 125 cm/hr were examined. The flow
rate did affect product recovery but had no impact on product
quality as assessed by SEC (% monomer) and clearance of HCP (Table
10), justifying the broader range of 25 to 125 cm/hr. The target
flow rates for the Phenyl Sepharose manufacturing operation remain
as previously established at 75 cm/hr and 37.5 cm/hr for the
elution phase.
TABLE-US-00010 TABLE 10 Phenyl sepharose flow rate evaluation Flow
rate Load (g protein/L % HCP (cm/hr) resin) % Recovery.sup.a %
Monomer.sup.b clearance 25 32.5 69 99.98 92.1 75 32.5 84 99.98 92.2
125 32.5 84 99.98 91.8 .sup.aPhenyl Sepharose step yield action
limit: .gtoreq.48% .sup.bPhenyl Sepharose step SEC action limit:
.gtoreq.98% monomer
[0630] The acceptable operating ranges for Phenyl Sepharose HP
chromatography were investigated. Hydrophobic interaction
chromatography can be affected by protein loading, ionic strength
(conductivity), and linear velocity. Protein loading can affect
selectivity and yield. Ionic strength of the load sample can affect
binding capacity, selectivity and resolution. Linear velocity may
affect mass transport properties, potentially resulting in
decreased resolution of process related impurities at very high
flow rates and axial dispersion at very low flow rates. The linear
flow rate range is expanded to 25 to 125 cm/hr. The other
acceptable operating ranges for Phenyl Sepharose chromatography are
unchanged from those previously established for the 6000 L process
and are listed in Table 10.
[0631] Comparable performance of the fine purification operations
in both processes was demonstrated. Changes introduced as part of
improved process B include: adjustment of the pH and conductivity
of the viral-inactivated pool, which serves as the Q Sepharose FF
load, to match the Q Sepharose equilibration buffer, limiting the Q
Sepharose load to less than 40 g protein per liter resin, and
elimination of the requirement to fractionate the Phenyl Sepharose
eluate at loads of less than 35 g protein per liter resin. The
quality of intermediates, as determined by SEC and WCX-10 assays,
were comparable between the two processes.
[0632] Typical elution profiles for the Q Sepharose FF and Phenyl
Sepharose HP chromatography steps for each process are provided in
FIGS. 2 and 3, respectively. The Q Sepharose flow-through
comprising adalimumab and was collected. The load volume amounts
for process B were higher than the previous process, due to the
greater loads at the previous chromatography step (Fractogel S) and
increased dilution volume; therefore the total flow-through volume
is correspondingly greater.
[0633] In sum, a requirement to fractionate the Phenyl Sepharose
eluate for loads less than 35 g protein per liter resin was
eliminated due to improvements in impurity clearance resulting from
the changes in the Fractogel S and Q Sepharose operations. In
addition, the linear flow rate range was expanded to 25 to 125
cm/hr.
Reduction in HCP
[0634] Process B included modifications to the Fractogel S and Q
Sepharose chromatography steps which were implemented to improve
control of process-related impurities such as host cell protein
(HCP) and, specifically, procathepsin L. A study was undertaken to
assess the impact of process B on the removal of these impurities.
The capacity of the Fractogel S, Q Sepharose FF and Phenyl
Sepharose HP columns to remove CHO host cell proteins was evaluated
at manufacturing scale. Host cell protein levels were determined by
HCP ELISA (see Example 3) and data are expressed in ng HCP/mg
adalimumab.
[0635] Representative samples were taken during process B and
assayed for HCP. The results are presented in Table 11. Changes to
the chromatography steps represent more rigorous chromatographic
conditions which would be expected to improve the HCP clearance.
Delipid filtration results reported are those from process A. The
delipid filtration step was unchanged process B, therefore the HCP
reduction factor achieved at this step is included in the overall
performance of process B. On average, process B is able to remove
greater than 4.35 log.sub.10 of HCP. Both Fractogel S
chromatography and Q Sepharose FF chromatography cleared more than
1 log.sub.10 HCP, and the depth filtration step also cleared more
then 1 log.sub.10. Additional HCP was removed by the Phenyl
Sepharose column, however, the clearance value was not calculable
because both the load and eluate HCP levels were below the level of
quantitation. The drug substance produced by process B exhibited
HCP levels below the limit of quantitation (LOQ) for the three
validation lots.
TABLE-US-00011 TABLE 11 Host cell protein clearance HCP in HCP out
Log.sub.10 reduction Chromatography step (ng/mg ada) (ng/mg ada)
factor Fractogel SO.sub.3- 1.71 column (average).sup.a Lot D
1,035,101 18,199 1.75 Lot E 747,748 16,079 1.67 Lot F 1,350,632
26,772 1.70 Delipid Filtration 1.58 (average).sup.b Lot 18,174
1,466 1.11 Lot H 34,369 805 1.63 Lot I 38,453 570 1.83 Lot J 25,774
466 1.74 Q Seph. FF 1.07 column (average).sup.a Lot D 269.98 Cycle
A: 28.41 0.98 Cycle B: 30.54 0.95 Lot E 313.44 Cycle A: 28.94 1.03
Cycle B: 29.82 1.02 Lot F 391.96 Cycle A: 22.51 1.24 Cycle B: 26.52
1.17 Phenyl Seph. N/A HP column (average).sup.a Lot D <40.44
<9.08 N/A Lot E <43.56 <8.65 N/A Lot F <44.65 <9.22
N/A Total Clearance.sup.c 4.35 .sup.aData from process B .sup.bData
from process A .sup.cLog 10 reduction factors less than 1 are not
included in the overall clearance calculation
[0636] Overall improvements in HCP and procathepsin L levels are
also shown in Tables 12 and 13, respectively, where process B
showed significant decreases in both levels in comparison to
process A.
[0637] Procathepsin L Process Mapping
[0638] Process intermediate samples were taken at several steps and
analyzed for fluorescence generated by activation of procathepsin L
to cathepsin L. Results are shown in Table 14 below for process B
and process A samples. The Fractogel S load and Phenyl Sepharose
load and eluate samples could not be evaluated due to interference
with the method. The Q Sepharose FF chromatography step has the
capability of removing greater than 90% of the detectable enzyme in
the load. The Q Sepharose flow-through and wash (FTW) from the
improved process contains approximately 50% less activatable
procathepsin L than the Q Sepharose FTW from the 6000 L previous
process. Reductions also occur between the Fractogel S and the Q
Sepharose steps during which the delipid filtration, concentration
by ultrafiltration, low pH viral inactivation and depth filtration
operations are performed.
TABLE-US-00012 TABLE 14 Procathepsin L mapping of the process A and
process B Process B (RFU/s/mg) Average .+-. Reduction Sample Lot S
Lot U Lot T SD Factor.sup.a Fractogel Eluate 46 59 57 54 .+-. 7 N/A
Pool Q Sepharose load 32 31 39 34 .+-. 4 1.6 Q Sepharose FTW 2.2
3.6 2.3 2.7 .+-. 0.8 13.4 Drug substance 2.6 3.0 2.7 2.8 .+-. 0.2
None Process A Lot Average .+-. Reduction Lot V W Lot X SD Factor
Fractogel Eluate 96 83 96 91 .+-. 7 N/A Pool Q Sepharose load 46 67
58 57 .+-. 10 1.6 Q Sepharose FTW 5.8 5.7 5.0 5.5 .+-. 0.5 10.5
Drug substance 4.2 3.9 3.8 4.0 .+-. 0.2 1.4 .sup.aThe reduction
factor is calculated using pre-rounded data for each lot and the
average of the three runs is reported.
The comparison of procathepsin L reduction in processes A and B is
displayed in FIG. 4. Process B exhibits lower procathepsin L levels
than process A at each intermediate step, indicating that the
modifications to the Fractogel S and Q Sepharose chromatography
steps improve process performance with respect to removal of this
impurity.
HCP Process Mapping
[0639] Process intermediate samples were collected from both
processes A and B, and analyzed for HCP content. This study was
performed in order to directly compare the two processes for HCP
reduction. The results of the HCP analysis are shown in Table 15.
Significant removal of HCP occurs at the Fractogel S and Q
Sepharose steps in both processes but process B exhibits improved
HCP clearance across both of these steps. The improved Fractogel S
step, which includes the second wash step prior to product elution,
has a reduction factor of 96 (1.96 log.sub.10) whereas the same
step in the previous process yields a reduction factor of 48 (1.67
log.sub.10). Both processes exhibit a reduction factor of 50
accomplished by the delipid filtration, performed between the
Fractogel S and Q Sepharose chromatography steps. The Q Sepharose
operation in process B is performed with the load adjusted to the
pH and conductivity of the column equilibration buffer. The HCP
reduction factor achieved by the improved Q Sepharose step is
four-fold greater than that demonstrated by the previous process
(21 vs. 5). Further reduction occurs across the Phenyl Sepharose
step such that the level of HCP is below the level of quantitation
in the improved process UF/DF pool and drug substance; the previous
drug substance samples exhibit very low but measurable levels of
HCP.
TABLE-US-00013 TABLE 15 Host cell protein mapping of processes A
and B Process B lots (ng HCP/mg adalimumab) Average .+-. Reduction
Sample Lot D Lot E Lot F SD Factor Filtered Harvest 1,330,000
813,000 2,130,000 1,420,000 .+-. 661,000 N/A Fractogel Eluate Pool
12,400 19,200 15,300 15,600 .+-. 3370 96 Q Sepharose load 554 220
371 382 .+-. 167 50 Q Sepharose FTW 18.5 20 17 18.5 .+-. 1.5 21
Drug substance.sup.a <5 <5 <5 <5 >4 Process A lots
(ng HCP/mg adalimumab) Average .+-. Reduction Lot V Lot W Lot X SD
Factor Filtered Harvest 2,030,000 2,520,000 1,870,000 2,140,000
.+-. 339,000 N/A Fractogel Eluate Pool 40,400 40,700 56,400 45,800
.+-. 9160 48 Q Sepharose load 536 1347 1248 1040 .+-. 442 50 Q
Sepharose FTW 98 213 283 198 .+-. 93 5 Drug substance 5 8 11 8 .+-.
3 24 .sup.aAll improved lot samples for this step were below the 5
ng/mg limit of quantitation. A value of 5 ng/mg was used to
estimate the reduction factor.
The comparison of HCP reduction in process B versus process A,
plotted on a log.sub.10 scale, is displayed in FIG. 5. Process B
exhibits lower HCP levels than process A at each intermediate step,
including a 10-fold difference following the Q Sepharose step,
indicating that the modifications to the Fractogel S and Q
Sepharose chromatography steps improve process performance with
respect to removal of HCPs.
Impact of the Capture and Fine Purification Operations on
Processing Capacity
[0640] Two changes were introduced to increase the processing
capacity of the capture and fine purification operations in process
B. The first was the increase in the allowable load limit on the
Fractogel S column from 30 g protein/L resin to 35 g protein/L
resin at pH 7 and from 30 g protein/L resin to 70 g protein/L resin
at pH 5. These changes allowed all of the filtered harvest material
from the bioreactor to be loaded onto the Fractogel S column. The
average load onto the Fractogel S column was approximately 9%
higher in the improved process (at pH 7) than the load in the
previous process (Table 16).
[0641] The second change was the removal of the requirement to
fractionate the Phenyl Sepharose product peak with loads of less
than 35 g protein/L resin. The fractionation resulted in discarding
a significant portion of the product peak in order to adequately
control host cell proteins. The changes implemented at the
Fractogel S and Q Sepharose steps to control host cell proteins and
procathepsin L levels rendered the fractionation of the Phenyl
Sepharose peak unnecessary. This change allowed running three
cycles of the Phenyl Sepharose column at a lower load range for
process B resulting in a 12% increase in total load on the Phenyl
Sepharose column compared with process A.
[0642] Table 16 compares the loads on the Fractogel S and the
Phenyl Sepharose columns as well as the final drug substance
amounts from the improved and previous processes. The improved
process exhibits an approximate 8% overall increase in adalimumab
yield for the three validation batches.
TABLE-US-00014 TABLE 16 Comparison of Fractogel S and Phenyl
Sepharose column loads and drug substance yields in processes A and
B Fractogel S Phenyl Sepharose Drug substance Process load.sup.a
load.sup.a yield.sup.a Process A (n = 15) 7641 .+-. 138 5947 .+-.
28 5290 .+-. 158 Process B (n = 3) 8375 .+-. 293 6752 .+-. 38 5748
.+-. 75 Increase in process B 9% 12% 8% .sup.aLoad and yield are
expressed in grams of protein
[0643] The improved method of purifying the antibody adalimumab
improved clearance of HCP and procathepsin L (relative to process
A), resulting in reduced levels in the drug substance. More
specifically, in a comparison of drug substance lot release data,
the following levels of HCP and procathepsin L were determined, as
described in Table 17.
TABLE-US-00015 TABLE 17 Comparison of HCP and procathepsin L in
processes A and B Lot release Process Process Process Process
Process Assay specification A.1 A.2 A3 A.4 B Host cell .ltoreq.70
ng/mg 46 .+-. 15 6 .+-. 3.sup.b 22 .+-. 19.sup. 9 .+-. 4 <5
protein (HCP) Procathepsin L .ltoreq.5% 18 .+-. 8.sup.a
<3.85.sup.c 65 .+-. 23.sup.za <3.61.sup.d <3.3.sup.e
.sup.aProcathepsin L specification does not apply to process A.1;
values provided for information only. .sup.b14 of 17 lots below LOQ
limit of 5 ng/mg; a value of 5 ng/mg used to calculate average and
standard deviation. .sup.cLOQ ranged from 3.30 to 3.85. .sup.dLOQ
ranged from 3.29 to 3.61. .sup.eLOQ was 3.3 (LOQ = Limit of
quantitation)
[0644] Extended characterization of the drug substance produced
using the process B was performed. Drug substance from the three
validation lots was analyzed and compared with an adalimumab
reference standard, using the assays including amino acid analysis,
circular dichroism, analytical centrifugation, QSTAR LC-mass
spectrometry, non-reduced tryptic and LYS C peptide mapping with MS
detection, free sulfhydryl assay, tryptic peptide mapping with MS
detection, immunoblot, L929 bioassay, and BIAcore. All batches of
drug substance manufactured by the improved process met the
acceptance criteria and are comparable to the reference
standard.
[0645] In sum, the performance of the process B has been
demonstrated to be comparable to the process A at fermentation,
capture and fine purification stages. Process B, however, exhibits
improved capability with regard to reduction of host cell protein
and procathepsin L, as well as an increase in capacity with regard
to adalimumab yield. Drug substance release testing and extended
characterization studies further demonstrate the comparability of
the adalimumab drug substance produced by process B with that
produced by process A.
TABLE-US-00016 TABLE 12 Overall improvement of HCP levels. Sample
description Process A.1 (3 K) Process A.2 (2 K) A B C D E F 1 Post
depth filtration 853,852 1,181,845 936,390 1,238,297 991,390
1,018,529 2 Fractogel-S eluate 6,739 15,772 16,286 17,528 15141
15426 3 Conc. Fractogel-S eluate 5980 13958 15361 14984 12769 434*
G H I J K L 4 Viral inactivated filtrate 2702 5074 5181 3826 3321
216 5 Q Seph. FF flow through 415 891 562 311 157 30 6 Final UF/DF
retentate 36 83 43 20 7 <LOQ HCP Q reduction (fold) 6.51 5.69
9.22 12.30 21.15 .sup. 7.20 Sample ID Sample descrition Process A.3
(6 K) Process A.4 (6 K) M N O P Q R 7 Post depth filtration
2,039,630 2,150,284 2,125,986 2,026,000 2,517,074 1,867,919 8
Fractogel-S eluate 35,223 31,461 46,072 40,399 40,710 56,415 Y Z
A.A A.B A.C A.D 9 Conc. Fractogel-S eluate 1157 NA NA .sup. 2,325
2,553 2,437 10 Viral inactivated filtrate 468 527 1,563 536 1,347
1,248 11 Q Seph. FF flow through 229 314 594 .sup. 98 213 283 12
Final UF/DF retentate 23 33 48 .sup. 5* 8 11 HCP Q reduction (fold)
2.04 1.68 2.63 .sup. 5.47 6.32 4.41 Sample ID Sample descrition
Process B.1 (6 K) Process B.2 (12 K) S T U V W X 7 Post depth
filtration 1,333,900 813,256 2,126,449 1,271,211 1,261,889
1,056,935 8 Fractogel-S eluate 12,430 19,150 15,257 9,558 13,130
17,427 A.E A.F A.G 9 Conc. Fractogel-S eluate N/A N/A N/A 4824 771
526 10 Viral inactivated filtrate 554 220 371 1,317 376 255 11 Q
Seph. FF flow through 18.5 20 17 14 3 0 12 Final UF/DF retentate
0.5 0 0 0 0 0 HCP Q reduction (fold) *lot was operated with delipid
filter in the process.
TABLE-US-00017 TABLE 13 Overall improvements in procathepsin L
levels. Sample ID Sample descrition Process A.1 (3 K) Process A.2
(3 K) A B C D E F 1 Fractogel-S eluate 54.07 51.23 61.04 42.19
50.14 48.28 2 Conc. Fractogel-S eluate 45.76 42.95 48.44 46.72
43.77 46.13 G H I J K L 3 Viral inactivated filtrate 41.54 36.37
43.70 36.49 38.28 38.94 4 Q Seph. FF flow through 6.55 6.22 7.16
3.39 3.37 3.41 5 Phenyl Seph FF eluate 6.82 6.92 8.75 2.57 2.11
3.01 6 Final UF/DF retentate 9.85 9.77 9.69 1.84 2.93 3.00
Cathepsin L Activity Q 6.34 5.85 6.10 10.76 11.36 11.42 reduction
(fold) Sample ID Sample descrition Process A.3 (6 K) Process A.4 (6
K) Process B.1 (6 K) Process B.2 (12 K) M N O P Q R S T U V W X 7
Fractogel-S eluate 98.26 77.10 106.41 97.42 84.91 94.14 45.78 58.55
56.61 26.70 40.70 41.05 Conc. Fractogel-S eluate 61.94 57.20 47.20
22.20 42.80 35.53 Y Z A.A A.B A.C A.D A.E A.F A.G 8 Viral
inactivated filtrate 51.49 64.38 69.29 46.43 67.28 57.59 31.85
31.19 38.56 13.20 45.90 42.32 9 Q Seph. FF flow through 10.02 9.86
10.05 5.81 5.65 4.96 2.21 3.57 2.25 0.40 1.30 0.9 10 Phenyl Seph FF
eluate 8.48 8.79 9.21 3.29 4.28 3.48 1.00 1.30 0.85 11 Final UF/DF
retentate 6.68 8.76 8.54 3.28 3.46 3.71 2.59 3.03 2.65 1.30 0.60
1.00 Cathepsin L Activity Q 5.14 6.53 6.89 7.99 11.91 11.61 14.41
8.74 17.14 33.00 35.31 47.02 reduction (fold) Cathepsin L
activitity: The unit used in red numbers is the fluorescent signal
release rate described as RFU/sec./mg D2E7
Example 3
Assay for HCP Detection
[0646] The following example describes an HCP ELISA method for the
determination of the residual Host Cell Protein (HCP) concentration
in adalimumab drug substance samples obtained from process B,
described in Example 2. Enzyme Linked Immunosorbent Assay (ELISA)
was used to sandwich the sample comprising the HCP antigen between
two layers of specific antibodies. This was followed by the
blocking of non-specific sites with Casein. The sample was then
incubated during which time the antigen molecules were captured by
the first antibody (coating antibody Cygnus goat anti-CHO (Chinese
Hamster Ovary), affinity purified). A second antibody (anti-CHO
host cell protein biotinylated) was then added which fixed to the
antigen (CHO host cell proteins) Importantly, the second antibody
specific to the HCPs was produced from the cells used to generate
the antibody. Neutravidin HRP-conjugated was added which binds to
the biotinylated anti-CHO host cell protein. This was followed by
the addition of K blue substrate. The chromogenic substrate was
hydrolyzed by the bound enzyme conjugated antibody, producing a
blue color. The reaction was stopped with 2 M H.sub.3PO.sub.4,
changing color to yellow. Color intensity was directly proportional
to the amount of antigen bound to the well. The HCP ELISA showed
improvements for determining HCP levels in an antibody preparation
than standard ELISA methods.
Example 4
Cathepsin L Kinetic Assay
[0647] A kinetic assay was developed and used to quantify cathepsin
L activity for adalimumab manufacturing process intermediates of
process B (see Example 2). The weak anion exchange HPLC assay
(WAX-10 HPLC) used to measure HCP for drug substance release
testing could not be used for this study since the variable protein
content and buffer composition of the in-process samples may
interfere with the method. The inability to directly quantitate
procathepsin L in the process intermediates led to the development
of an assay which measured the activity of cathepsin L by a kinetic
fluorescence method. The kinetic assay, i.e., a high throughput
fluorescent enzymatic method, has less interference for in-process
samples than standard methods used to detect procathepsin L levels.
The kinetic assay also provides a means for examining the
reliability of the process for purifying adalimumab in-process
samples described in Examples 1 and 2.
[0648] This method forces the activation of the procathepsin L in
the samples to cathepsin L by addition of dextran sulfate. A
fluorogenic peptide substrate, Z-leucine-arginine-AMC
(7-amino-4-methyl coumarin), was used to detect cathepsin L
activity at excitation 380 nm and emission 460 nm. The level of
fluorescence activity in the samples was determined by the slope of
the fluorogenic signal generated by the cleavage of the substrate
per second. The range of this fluorescent activity assay was
determined to be between 0.0144 to 1.04 RFU/sec. This activity was
correlated to the amount of adalimumab present in the test sample;
hence results are report as RFU/sec/mg adalimumab. Optimum
activation conditions to achieve the maximum fluorescent signal
were developed for each process intermediate sample using JMP
software derived DOE experiments. The recommended activation
conditions for this assay are summarized in Table 16.
Materials and Methods
[0649] Preparation of 500 mM DTT Stock Solution
[0650] 7.7 grams of Ultrapure DTT (Invitrogen) was added into 90 mL
of Milli-Q water and mixed until homogenous. The solution was
topped up the solution with Milli-Q Water to a final volume of 100
mL. This 500 mM DTT stock was then aliquoted and stored at
-80.degree. C.
[0651] Preparation of the Activation Buffer (25 mM NaOAc, 5 mM DTT,
1 mM EDTA pH 5.5)
[0652] 3.44 grams of sodium acetate (J. T. Baker), 0.38 grams of
EDTA (J. T. Baker) and 950 mL Milli-Q water were added to a proper
container and mixed until completely homogenous. The pH of the
buffer was adjusted to 5.5 with 1 M HCl, and brought up to the
final volume of 1 L in a volumetric flask. The buffer was filtered
through a 0.22 .mu.m filter and stored at 4.degree. C. prior to
use. 500 .mu.L of DTT stock solution (500 mM described above) was
added to 50 mL of buffer to a final concentration of 5 mM at the
day of use.
[0653] Preparation of Dextran Sulfate+0.1% Sodium Azide Stock
Solution
[0654] 1 gram of dextran sulfate (EM Science) was added into 90 mL
of Milli-Q water and mixed by until homogenous. 100 .mu.L sodium
azide was added from a 1 mg/mL stock solution (J. T. Baker). The
solution was topped up to a final volume of 100 mL. This solution
was then aliquoted and stored at -80.degree. C.
Kinetic Assay Set-Up
[0655] Samples to be tested for cathepsin L activity require
activation of the proenzyme (procathepsin L) to active enzyme
(cathepsin L). This was accomplished by diluting samples in
activation buffer, adding dextran sulfate and incubating at
37.degree. C. for an appropriate time (details discussed in below).
After activation, samples can be stored at -80.degree. C. and
remain stable. Optimal activation conditions determined for
in-process samples are shown in Table 18.
TABLE-US-00018 TABLE 18 Summary of refined activation conditions
for in-process samples Dextran sulfate Activation Sample Dilution
(.mu.g/mL) time (hr) Fractogel eluate 700 0.035 6 Q Sepharose Load
700 0.035 6 Q Sepharose FTW 70 0.035 18 Phenyl eluate 200 0.035 6
Drug substance 600 0.035 6
[0656] On the day of testing, an aliquot of the test samples were
removed from -80.degree. C. and thawed in an ice bath. Once the
test samples have thawed, (2.times.) 100 .mu.L of each sample was
loaded into a black polystyrene micro titer plate (Corning
cat#3650). An aliquot of the Z-L-R-AMC Fluorogenic Peptide
Substrate VII (R&D Systems) was thawed while protected from
light. The substrate was diluted 1:1350 with the acetate buffer to
a final concentration of 20 .mu.M. 100 .mu.L of the fluorogenic
substrate was added to each well. The plate was then mixed for
.about.1 second and incubated at 37.degree. C. for 3 minutes, while
protected from light. The plate was then placed in the fluorescent
plate reader that has been set to 37.degree. C. The excitation
wavelength was set to 380 nm and the emission was set to 460 nm.
The fluorescence of each well was measured every 3 minutes for 30
minutes and the rate of substrate hydrolysis was calculated. The
results, which take into consideration the dilution factor, were
then divided by the adalimumab concentration for comparison.
Results using this kinetic assay are described above in Example
2.
[0657] Adalimumab concentration was determined by A.sub.280 using
an extinction coefficient of 1.39. Adalimumab quantitation was
performed on study samples using Poros A analysis. Sample dilutions
were applied to achieve readings within the standard curve. A
Shimadzu HPLC system was configured with a Poros A ImmunoDetection
sensor cartridge (Applied Biosystems, Foster City, Calif.). The
column was maintained at ambient temperature. The system was run at
2 mL/minute. The auto sampler tray temperature was set at 4.degree.
C. Absorbance was monitored at 280 nm. Buffer A was lx PBS; buffer
B was 0.1 M acetic acid and 150 mM sodium chloride. The sample was
injected and Adalimumab was eluted using 100% buffer B.
[0658] The turnover of fluorogenic peptide using Fractogel load
(see first eluate Example 2; process B) from material obtained from
CHO cell expression of adalimumab is shown in FIG. 6. This sample
was diluted to 200, 50 and 20 .mu.g/mL of adalimumab with
activation buffer using 0.5 .mu.g/mL dextran sulfate, and incubated
at 37.degree. C. for 16 hours. This lot at 50 and 20 .mu.g/mL
showed linear responses. The R.sup.2 values are .gtoreq.0.99.
However, the lot at 200 .mu.g/mL shows nonlinear substrate turnover
towards the end of the 30 minutes measurement time, resulting in a
lower R.sup.2 value of 0.91. Therefore, careful sample dilution is
critical to maintain linear hydrolysis rates.
[0659] Assays were also performed to confirm that the kinetic assay
using cathepsin activity to determine the level of procathepsin A
were compliant with ICH guidelines, including precision analysis,
including repeatability precision. Furthermore, it was determined
that the type of container, e.g., glass and polypropylene vials
influences of cathepsin L activity. The results suggest that higher
levels of cathepsin L are achieved when incubating in a
polypropylene container as opposed to a glass container. In both
cases, the addition of 0.5 .mu.g/mL dextran sulfate was required
for procathepsin L activation at pH 5.5.
[0660] In sum, the precision of the kinetic assay demonstrates that
this assay is valid for detection of potential cathepsin L activity
of adalimumab process intermediates.
[0661] This application is related to U.S. Pat. Nos. 6,090,382,
6,258,562, and 6,509,015. This application is also related to U.S.
patent application Ser. No. 09/801,185, filed Mar. 7, 2001; U.S.
patent application Ser. No. 10/302,356, filed Nov. 22, 2002; U.S.
patent application Ser. No. 10/163,657, filed Jun. 5, 2002; and
U.S. patent application Ser. No. 10/133,715, filed Apr. 26, 2002;
U.S. patent application Ser. No. 10/222,140, filed Aug. 16, 2002;
U.S. patent application Ser. No. 10/693,233, filed Oct. 24, 2003;
U.S. patent application Ser. No. 10/622,932, filed Jul. 18, 2003;
U.S. patent application Ser. No. 10/623,039, filed Jul. 18, 2003;
U.S. patent application Ser. No. 10/623,076, filed Jul. 18, 2003;
U.S. patent application Ser. No. 10/623,065, filed Jul. 18, 2003;
U.S. patent application Ser. No. 10/622,928, filed Jul. 18, 2003;
U.S. patent application Ser. No. 10/623,075, filed Jul. 18, 2003;
U.S. patent application Ser. No. 10/623,035, filed Jul. 18, 2003;
U.S. patent application Ser. No. 10/622,683, filed Jul. 18, 2003;
U.S. patent application Ser. No. 10/622,205, filed Jul. 18, 2003;
U.S. patent application Ser. No. 10/622,210, filed Jul. 18, 2003;
and U.S. patent application Ser. No. 10/623,318, filed Jul. 18,
2003. This application is also related to PCT/US05/12007, filed
Apr. 11, 2005. The entire contents of each of these patents and
patent applications are hereby incorporated herein by
reference.
EQUIVALENTS
[0662] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims. The contents of all references, patents and
published patent applications cited throughout this application are
incorporated herein by reference.
Sequence CWU 1
1
371107PRTArtificial Sequenceadalimumab light chain variable region
1Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn
Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr
Cys Gln Arg Tyr Asn Arg Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys 100 105 2121PRTArtificial
Sequenceadalimumab heavy chain variable region 2Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25 30 Ala
Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40
45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val
50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser
Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala
Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr Ala Ser
Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser
Ser 115 120 39PRTArtificial Sequenceadalimumab light chain variable
region CDR3 3Gln Arg Tyr Asn Arg Ala Pro Tyr Xaa 1 5
412PRTArtificial Sequenceadalimumab heavy chain variable region
CDR3 4Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa 1 5 10
57PRTArtificial Sequenceadalimumab light chain variable region CDR2
5Ala Ala Ser Thr Leu Gln Ser 1 5 617PRTArtificial
Sequenceadalimumab heavy chain variable region CDR2 6Ala Ile Thr
Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu 1 5 10 15 Gly
711PRTArtificial Sequenceadalimumab light chain variable region
CDR1 7Arg Ala Ser Gln Gly Ile Arg Asn Tyr Leu Ala 1 5 10
85PRTArtificial Sequenceadalimumab heavy chain variable region CDR1
8Asp Tyr Ala Met His 1 5 9107PRTArtificial Sequence2SD4 light chain
variable region 9Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser
Ala Ser Ile Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser
Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro
Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Thr Leu
Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly
Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp
Val Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ser Ala Pro Tyr 85 90 95
Ala Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105
10121PRTArtificial Sequence2SD4 heavy chain variable region 10Gln
Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10
15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr
20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp
Trp Val 35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr
Ala Asp Ser Val 50 55 60 Glu Gly Arg Phe Ala Val Ser Arg Asp Asn
Ala Lys Asn Ala Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Pro
Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Lys Ala Ser Tyr Leu
Ser Thr Ser Ser Ser Leu Asp Asn Trp Gly 100 105 110 Gln Gly Thr Leu
Val Thr Val Ser Ser 115 120 119PRTArtificial Sequence2SD4 light
chain variable region CDR3 11Gln Lys Tyr Asn Ser Ala Pro Tyr Ala 1
5 129PRTArtificial SequenceEP B12 light chain variable region CDR3
12Gln Lys Tyr Asn Arg Ala Pro Tyr Ala 1 5 139PRTArtificial
SequenceVL10E4 light chain variable region CDR3 13Gln Lys Tyr Gln
Arg Ala Pro Tyr Thr 1 5 149PRTArtificial SequenceVL100A9 light
chain variable region CDR3 14Gln Lys Tyr Ser Ser Ala Pro Tyr Thr 1
5 159PRTArtificial SequenceVLL100D2 light chain variable region
CDR3 15Gln Lys Tyr Asn Ser Ala Pro Tyr Thr 1 5 169PRTArtificial
SequenceVLL0F4 light chain variable region CDR3 16Gln Lys Tyr Asn
Arg Ala Pro Tyr Thr 1 5 179PRTArtificial SequenceLOE5 light chain
variable region CDR3 17Gln Lys Tyr Asn Ser Ala Pro Tyr Tyr 1 5
189PRTArtificial SequenceVLLOG7 light chain variable region CDR3
18Gln Lys Tyr Asn Ser Ala Pro Tyr Asn 1 5 199PRTArtificial
SequenceVLLOG9 light chain variable region CDR3 19Gln Lys Tyr Thr
Ser Ala Pro Tyr Thr 1 5 209PRTArtificial SequenceVLLOH1 light chain
variable region CDR3 20Gln Lys Tyr Asn Arg Ala Pro Tyr Asn 1 5
219PRTArtificial SequenceVLLOH10 light chain variable region CDR3
21Gln Lys Tyr Asn Ser Ala Ala Tyr Ser 1 5 229PRTArtificial
SequenceVL1B7 light chain variable region CDR3 22Gln Gln Tyr Asn
Ser Ala Pro Asp Thr 1 5 239PRTArtificial SequenceVL1C1 light chain
variable region CDR3 23Gln Lys Tyr Asn Ser Asp Pro Tyr Thr 1 5
249PRTArtificial SequenceVL0.1F4 light chain variable region CDR3
24Gln Lys Tyr Ile Ser Ala Pro Tyr Thr 1 5 259PRTArtificial
SequenceVL0.1H8 light chain variable region CDR3 25Gln Lys Tyr Asn
Arg Pro Pro Tyr Thr 1 5 269PRTArtificial SequenceLOE7.A light chain
variable region CDR3 26Gln Arg Tyr Asn Arg Ala Pro Tyr Ala 1 5
2712PRTArtificial Sequence2SD4 heavy chain variable region CDR3
27Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asn 1 5 10
2812PRTArtificial SequenceVH1B11 heavy chain variable region CDR3
28Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Lys 1 5 10
2912PRTArtificial SequenceVH1D8 heavy chain variable region CDR3
29Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Tyr 1 5 10
3012PRTArtificial SequenceVH1A11 heavy chain variable region CDR3
30Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu Asp Asp 1 5 10
3112PRTArtificial SequenceVH1B12 heavy chain variable region CDR3
31Ala Ser Tyr Leu Ser Thr Ser Phe Ser Leu Asp Tyr 1 5 10
3212PRTArtificial SequenceVH1E4 heavy chain variable region CDR3
32Ala Ser Tyr Leu Ser Thr Ser Ser Ser Leu His Tyr 1 5 10
3312PRTArtificial SequenceVH1F6 heavy chain variable region CDR3
33Ala Ser Phe Leu Ser Thr Ser Ser Ser Leu Glu Tyr 1 5 10
3412PRTArtificial Sequence3C-H2 heavy chain variable region CDR3
34Ala Ser Tyr Leu Ser Thr Ala Ser Ser Leu Glu Tyr 1 5 10
3512PRTArtificial SequenceVH1-D2.N heavy chain variable region CDR3
35Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Asn 1 5 10
36321DNAArtificial Sequenceadalimumab light chain variable region
36gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtagggga cagagtcacc
60atcacttgtc gggcaagtca gggcatcaga aattacttag cctggtatca gcaaaaacca
120gggaaagccc ctaagctcct gatctatgct gcatccactt tgcaatcagg
ggtcccatct 180cggttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag cctacagcct 240gaagatgttg caacttatta ctgtcaaagg
tataaccgtg caccgtatac ttttggccag 300gggaccaagg tggaaatcaa a
32137363DNAArtificial Sequenceadalimumab heavy chain variable
region 37gaggtgcagc tggtggagtc tgggggaggc ttggtacagc ccggcaggtc
cctgagactc 60tcctgtgcgg cctctggatt cacctttgat gattatgcca tgcactgggt
ccggcaagct 120ccagggaagg gcctggaatg ggtctcagct atcacttgga
atagtggtca catagactat 180gcggactctg tggagggccg attcaccatc
tccagagaca acgccaagaa ctccctgtat 240ctgcaaatga acagtctgag
agctgaggat acggccgtat attactgtgc gaaagtctcg 300taccttagca
ccgcgtcctc ccttgactat tggggccaag gtaccctggt caccgtctcg 360agt
363
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